Identification of antigen or ligand-specific binding proteins

ABSTRACT

The present invention discloses novel methods for the generation, expression and screening of diverse collections of binding proteins such as antibodies or fragments thereof in vertebrate host cells in vitro, for the identification and isolation of ligand- or antigen-specific binding proteins. The methods disclosed herein allow the expression of diverse collections of binding proteins from at least one vector construct, which optionally can give rise to collections of diverse binding proteins upon transfer and expression into vertebrate host cells in situ.

The present application is a divisional application of U.S. patentapplication Ser. No. 12/397,957 filed Mar. 4, 2009, which claimspriority to U.S. Provisional Patent Application No. 61/125,886 file Apr.29, 2008 and European Patent Application No. 08004096.7 filed Mar. 5,2008. The entire contents of each of the above documents areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention discloses novel methods for the generation,expression and screening of diverse collections of binding proteins invertebrate cells in vitro, allowing the identification and isolation ofligand- or antigen-reactive binding proteins. In particular, the presentinvention relates to methods for the retroviral expression, isolationand identification of at least one nucleotide sequence encoding abinding protein such as an antibody or fragment thereof specific for adesired antigen or ligand.

BACKGROUND

Display technologies have played an important role in the isolation ofspecific high-affinity binding proteins for diagnostic and therapeuticapplications in a vast number of disorders and diseases. Thesetechnologies extend into the broad field of antibody engineering,synthetic enzymes, proteomics, and cell-free protein synthesis.Biomolecular display technologies, which allow the construction of alarge pool of modularly coded biomolecules, their display for propertyselection, and rapid characterisation (decoding) of their structures,are particularly useful for accessing and analyzing protein diversity ona large scale. Recently, in vitro display technologies have come toprominence due to the isolation of antibodies by phage display, ribosomedisplay and microbial display, which have now become mainstream antibodyand protein engineering platforms. However, microbial expression anddisplay systems suffer from limitations in particular for the expressionof large, dimeric vertebrate proteins, like antibodies. This is due tothe general inability to express full-length antibodies in suchexpression systems, which requires the display of engineered antibodyfragments, but also due to the lack of glycosylation, absence ofchaperone proteins, lack of subcellular compartments and eukaryotic cellspecific protein trafficking, that individually and collectively resultin protein folding artefacts in microbially expressed mammalianproteins. Recently, in vitro display methods have also been developedemploying eukaryotic host cells, including yeast, plants and mammaliancells. Yeast and plant cell expression systems also suffer from a lackof glycosylation and specific vertebrate and mammalian cell-specificchaperones, so that the same limitations with regard to protein foldingapply for the expression of vertebrate proteins in such systems.Expression, proper protein folding and posttranslational modification oflarge recombinant proteins, like antibodies, can only be expected tooccur with reasonable efficiency and quality in vertebrate expressionsystems, ideally expressing proteins in the phylogenetically mostclosely related cell system.

Therefore, therapeutically interesting proteins, like antibodies fromrodents or humans, are ideally expressed in rodent or human cells, andit is not surprising that only expression systems from such species areapproved by regulatory authorities for the production ofclinically-grade full-length therapeutic antibodies. However, vertebrateand mammalian cell based expression systems are laborious, requirelong-time frames to establish stably producing cell lines and clones,and an efficient and controlled genetic modification of such cells isoften not trivial and therefore makes these systems less attractive forscreening and display methods. For instance, DNA transfection methodscannot be controlled for the number of DNA constructs that are eithertransiently or stably incorporated into transfected cells, whichprecludes clonal expression of protein libraries and therefore a cleangene to phenotype screen. The alternative viral systems either lack aproper control of clonal expression, a stable maintenance of the geneticconstructs, and/or suffer from the fact that such systems often causecytopathic effects in the target cells (e.g. vaccinia virus expression),such that protein clones either cannot be displayed and/or sequentiallyenriched for a particular phenotype, like e.g. specific binding to anantigen.

It is thus an object of the present invention to provide a method thatclearly overcomes all of the above-mentioned limitations and drawbacksof prior art prokaryotic and eukaryotic gene expression and selectionsystems. The method according to the invention utilises stableretroviral expression of binding proteins such as, in particular,antibodies in mammalian cells, in particular B lymphocytic cell lines,such that stable and preferably clonal expression of antibody proteinsis achieved in the presence of proper glycosylation, chaperone proteinsand protein trafficking, ensuring proper protein folding and allowingefficient and, if desired, repeated screening for antigen-bindingantibody clones. Since the preferred embodiment of the method accordingto the invention is based on the retroviral expression of antibodies orfragments thereof in precursor lymphocytes the technology disclosedherein is termed ‘Retrocyte Display’ (for retroviral preB lymphocytedisplay).

SUMMARY OF THE INVENTION

The present invention generally relates to the provision of therapeuticor diagnostic antibodies or fragments thereof. In particular, it relatesto the identification and selection of antigen-reactive antibodies withfully human amino acid sequences that are of interest for therapeuticapplications. The embodiments of the invention involve retroviralexpression vectors enabling the expression of diverse collections ofbinding proteins, including antibodies or fragments thereof, invertebrate, preferably mammalian, cells and methods for the efficientisolation of ligand- or antigen-reactive molecules. The presentinvention provides novel methods for the generation of diversecollections of binding proteins, such as antibodies or fragmentsthereof, by three alternative methods. First, by chain shuffling of atleast one heavy or light chain molecule against a diverse collection(library) of light or heavy chains, (chain-shuffling approach), orsecond, by diversification of at least one combination of an antibodyheavy and light chain after retroviral transduction into vertebratecells in situ by somatic mutation of retrovirally transduced expressionconstructs (somatic mutation approach), or third, by V(D)J recombinationof retrovirally transduced expression constructs containing the codingregions for variable binding domains of antibodies in “quasi-germline”configuration, i.e. still separated into V, optionally D and J genesegments (V(D)J recombination approach). It is to be understood thatdiverse collections of binding proteins, including antibodies orfragments thereof, can also be generated by any combination of theabove-mentioned methods. Preferably, said binding proteins or antibodiesor fragments thereof are displayed on the surface of precursorlymphocytes.

The present invention particularly provides methods allowing the stable,and optionally clonal, expression of diverse collections of bindingproteins, preferably antibodies, in vertebrate cells using retroviraltransduction, which greatly facilitates the amplification, isolation,and cloning of binding protein encoding genes, in comparison toalternative, plasmid-based or non-integrating virus-based vertebrateexpression systems known in the art. As a representative but notlimiting example, the retroviral transduction of murine precursorlymphocytes that are incapable of expressing endogenous antibodies isdisclosed, such that only heterologous, recombinant antibodies areexpressed in the host cells as membrane-bound antibodies. Furthermore,the invention illustrates how cells that express ligand- orantigen-reactive binding proteins, such as antibodies or fragmentsthereof, can be isolated and optionally expanded in vitro, in order toiteratively enrich for a population of antigen-reactive binder cells,from which genes encoding antigen- or ligand-reactive binding proteinscan subsequently be cloned and sequenced by standard molecular biologyprocedures known in the art (FIG. 1).

Although a preferred embodiment of the method according to the inventionis directed to the retroviral expression of binding proteins, preferablyhuman, full-length antibodies, it can likewise be used for theexpression of any fragment thereof (e.g. single chain F_(v) or F_(ab)fragments of antibodies). Retroviral transduction protocols aredisclosed which optionally allow (i) delivery of single binding proteinencoding constructs into single target cells, in order to ensure clonalexpression of binding proteins in the host cells; (ii) shuffling of atleast one expression construct encoding a first polypeptide chain withat least one expression construct encoding a second polypeptide chain,thereby generating a functional multimeric binding protein (e.g. anantibody molecule); (iii) somatic mutation of at least one expressionconstruct encoding at least one binding protein upon transduction ofvertebrate cells in situ; and (iv) generation of binding proteinexpression from at least one expression construct by the mechanism ofV(D)J recombination upon retroviral transduction into vertebrate cellsin situ.

In order to achieve somatic mutation of binding protein encodingconstructs in situ, retroviral expression vectors and their utilizationare disclosed, wherein said vectors contain cis-regulatory geneticelements targeting somatic hypermutation to protein encoding sequences,preferably via an activation-induced cytidine deaminase (AID) pathway(Papavasiliou & Schatz, 2002), or by using other enzymes targetingsomatic mutations to binding protein encoding sequences. For thegeneration of diverse collections of binding proteins, preferablyantibodies or fragments thereof, by V(D)J recombination in situ,retroviral vector constructs and their utilization are disclosed,wherein said constructs contain variable (V), optionally diversity (D),and joining (J) gene segments arranged in “quasi-germline” configurationallowing assembly of coding regions for immunoglobulin orimmunoglobulin-like binding proteins via recombination activating gene(RAG)-mediated rearrangement of the gene segments by the process knownas V(D)J recombination (Grawunder et al., 1998).

According to a further aspect, the present invention further illustrateshow retrovirally transduced cells stably expressing diverse collectionsof recombinant binding proteins are subsequently labelled by binding toat least one ligand or antigen of interest, and how cells binding to theaforementioned ligand or antigen of interest are detected by appropriatesecondary reagents. Methods for the specific labelling of ligand- orantigen-reactive cells and their enrichment or isolation, preferably byhigh-speed fluorescent activated cell sorting (FACS), are described. Dueto the stable expression phenotype of retrovirally transduced cells, itis described how antigen-reactive cells may optionally be isolated andagain expanded in tissue culture, such that optionally iterative cyclesof antigen labelling, antigen-directed enrichment, and expansion ofligand or antigen-reactive cells can be performed, until subcloning ofthe cells is performed allowing the identification of the nucleotidecoding region for antigen-reactive antibodies by standard PCR cloningmethods (FIG. 1).

The methods disclosed herein allow the expression of diverse collectionsof antibody chains or fragments thereof from at least one vectorconstruct, which optionally can give rise to collections of diversebinding proteins upon transfer and expression into vertebrate cells insitu. Expression of antibody chains in vertebrate cells is preferablymediated by retroviral transduction.

As such, a first aspect of the present invention refers to a method forthe isolation and identification of at least one nucleotide sequenceencoding an antibody or fragment thereof specific for a desired antigenor ligand, comprising the steps of:

(a) transducing at least one retroviral expression construct encoding anantibody or fragment thereof into vertebrate host cells;

(b) expressing said antibody or fragment thereof in said vertebrate hostcells;

(c) enriching vertebrate host cells expressing said antibody or fragmentthereof on the basis of its ability to bind to said desired antigen orligand; and

(d) isolating and identifying said at least one nucleotide sequenceencoding said antibody or fragment thereof from the retrovirallytransduced and enriched vertebrate host cells.

In addition to the aforementioned steps, step (d) may be preceded by astep of expanding the enriched vertebrate host cells in tissue culture.Furthermore, step (c) may be followed by a step of expanding theenriched vertebrate host cells in tissue culture, after which step (c)is repeated at least once before step (d) is carried out.

To achieve clonal expression of at least one antibody it is preferableto control the retroviral transduction such that the majority ofretrovirally transduced cells are genetically modified by only onerecombinant retroviral construct per antibody chain integrating in tothe host cell genome. Therefore, in one embodiment of the presentinvention, retroviral transduction is performed at a multiplicity ofinfection (MOI) of equal to or less than 0.1.

An antibody according to a method of the present invention is preferablya full-length antibody. A fragment of an antibody may be selected fromthe group consisting of: a heavy chain, a light chain, a single V_(H)domain, a single V_(L) domain, a scFv fragment, a Fab fragment, and aF(ab′)2 fragment. The antibody or fragment(s) thereof may have anaturally occurring amino acid sequence, an artificially engineeredamino acid sequence or a combination thereof.

Whilst the method of the present invention is used preferably for theisolation and identification of at least one nucleotide sequenceencoding an antibody chain, it would be apparent to a person skilled inthe art that the method of the present invention can also be used forthe isolation and identification of at least one nucleotide sequenceencoding any monomeric or multimeric cell surface receptor belonging tothe Ig-superfamily, and any functional fragment thereof, or a monomericor multimeric cell surface receptor belonging to the TNFα-receptorsuperfamily, or any fragment thereof.

Furthermore, where the binding protein is a full-length antibody, thefull-length antibody is selected from the group consisting of a fullyhuman antibody, a humanized antibody, in which CDR regions of anon-human antibody or antibodies have been grafted onto a human antibodyframework, and a chimeric antibody, in which variable region domainsfrom one vertebrate species are combined with constant region domains ofanother vertebrate species, with the constant domain of the chimericantibody preferably being derived from a human antibody or antibodies.

In an embodiment of the methods disclosed herein, the vertebrate hostcells may be derived from a group of species comprising cartilaginousfish, bony fish, amphibians, reptilia, birds and mammals. The group ofspecies of mammals may include pigs, sheep, cattle, horses and rodents.The group of rodents may further comprise mice, rats, rabbits and guineapigs. In a preferred embodiment of the present invention, the vertebratehost cell species is mouse (Mus musculus).

The vertebrate host cells for use in a method of the present inventioncan be derived from any vertebrate organ, but are preferably derivedfrom lymphocyte lineage cells. The preferred lymphocytes for use in thepresent invention are of the B cell lineage, because these cells expressantibody-specific chaperone proteins, and because accessory molecules,like Igα and Igβ required to mediate cell surface anchoring ofantibodies are expressed in these cells. More preferably, the B cellsare precursor B lymphocytes, as preB cells can be found that do notexpress any endogenous antibody chains. In fact, the preferredlymphocytes as utilised in the present invention are unable to expressendogenous antibody polypeptides including components of the so-calledsurrogate light chain, encoded by the genes lambda-5, VpreB1 and VpreB2.Therefore, the preferred lymphocytes express accessory membrane proteinsfacilitating membrane deposition of antibody molecules, such as the Bcell specific Igα and Igβ molecules, but they lack expression of anyendogenous antibody polypeptide or surrogate light chain component.However, it shall be noted that it may be possible to express Igα andIgβ molecules ectopically, by methods known in the art, e.g. stabletransfection with expression vectors for these proteins. In a preferredembodiment of the present invention, antibody molecules are anchored tothe cell membrane of lymphocytes via endogenously expressed Igα and Igβproteins, which are naturally expressed in murine pre-B lymphocytes.

The methods disclosed herein include procedures allowing the isolationof cells displaying desired binding characteristics for a ligand orantigen of interest and the isolation of genes encoding a desiredbinding protein of interest. The preferred method of retroviralexpression of an antibody in vertebrate cells disclosed herein allowsfor stable and preferably clonal expression of antibodies, which greatlyfacilitates the amplification, isolation, and cloning of antibodyencoding genes, in comparison to alternative, plasmid-based ornon-integrating virus-based vertebrate expression systems known in theart. The disclosed methods allow for efficient generation of diversecollections of binding proteins in vitro by either:

(i) shuffling of at least one expression construct encoding at least onepolypeptide chain of a multimeric binding protein (like e.g. a heavychain of an antibody), with at least one expression construct encodingat least one matching polypeptide chain (like e.g. a light chain of anantibody) generating a functional multimeric binding protein (like e.g.an antibody);(ii) somatic mutation of at least one expression vector encoding atleast one binding protein upon transfer into vertebrate cells in situ;(iii) somatic recombination of V (variable), optionally D (diversity),and J (joining) gene segments encoding variable binding domains ofimmunoglobulins and immunoglobulin-like molecules contained in at leastone expression vector upon transfer into vertebrate cells in situ, bythe process known as V(D)J recombination; or(iv) by any combination of procedures (i), (ii), and (iii).

According to a preferred embodiment, the at least one nucleotidesequence is a plurality of nucleotide sequences that comprise anantibody heavy chain sequence and multiple antibody light chainsequences, or—in the alternative—comprise an antibody light chainsequence and multiple antibody heavy chain sequences.

According to another preferred embodiment, the antibody or fragmentthereof comprises a variable binding domain encoded by the at least oneretroviral expression construct enabling V(D)J recombination in order togenerate a coding sequence for a variable binding domain upon retroviraltransduction or

In a further preferred embodiment, step (b) of the above method isperformed under mutagenizing conditions, preferably via the expressionof activation induced cytidine deaminase (AID) which is eitherendogenously or ectopically expressed, wherein the ectopic expression ofAID is performed under inducible conditions.

In one aspect of the above method, the at least one retroviralexpression construct encoding said antibody or fragment thereof containsa combination of cis-regulatory promoter and enhancer elements allowingthe targeting of AID mediated somatic mutation to a variable bindingdomain encoded by the expression construct, wherein the promoter andenhancer elements are selected from the group consisting of

(a) immunoglobulin heavy chain promoter, intron enhancer (EμH) and 3′αenhancer elements,

(b) immunoglobulin κ light chain promoter, κ intron enhancer (κiE) and3′κ enhancer (3′κE) elements,

(c) immunoglobulin λ light chain promoter, λ2-4 and λ3-1 enhancerelements, and

(d) any functional combination thereof.

DESCRIPTION OF THE FIGURES

FIG. 1: This figure illustrates the principle of ‘Retrocyte Display’allowing the identification and isolation of a binding protein such asan antibody, specific for a desired antigen or ligand. In a first step,at least one retroviral expression construct that can give rise toexpression of a diverse collection of binding proteins is stablytransduced into suitable vertebrate host cells (“selector cells”). Thisis accomplished by transfecting at least one retroviral vector encodingat least one binding protein into retroviral packaging cells (step 1),which may either constitutively or transiently express retroviralproteins Gag, Pol and Env. Packaging cells transfected with the at leastone retroviral binding protein construct will then produce recombinantretroviral particles within 24-72 hours post transfection, containingthe at least one retroviral expression construct. The resultingretroviral particles accumulate in the cell culture supernatant of theretroviral packaging cells, and can be used to transduce suitablevertebrate host cells (“selector cells”) (step 2), which then expressthe binding protein. In the preferred method, the binding proteins suchas antibodies or fragments thereof are expressed on the cell surface ofthe “selector cells” and the cells then are labelled with a desiredantigen or ligand (step 3). Antigen- or ligand binding cells are thenpreferably analyzed by fluorescent activated cell sorting (FACS) andcells that exhibit specific antigen binding, are separated from thenon-binding cell population preferably by preparative, high-speed FACS(step 4). Antigen- or ligand reactive cells may optionally be expandedin tissue culture again, and due to the stable expression phenotype ofretrovirally transduced cells, cycles of antigen-directed cell sortingand tissue culture expansion may be repeated, up to the point that adetectable antigen- or ligand reactive cell population is obtained. Thisantigen- or ligand reactive cell population may be subjected to a final,preferable, single-cell sorting step, or may directly be used forcloning of binding protein encoding genes on a population basis. In thenext step (step 5), the coding regions of relevant binding domains arecloned from the antigen- or ligand-selected cell pools or cell clones,by RT-PCR or genomic PCR using primer pairs binding to sequencesspecific for the binding protein library and/or specific for othervector sequences, by standard methods known in the art. Cloned andsequenced coding regions for binding proteins may then optionally beexpressed as recombinant proteins in any expression system of choice forfurther functional characterization and to confirm antigen- or ligandbinding specificity (step 6).

FIG. 2: (a) This figure illustrates the schematic structure ofantibodies or immunoglobulins and fragments thereof, which are thepreferred binding proteins according to the disclosed invention. FIG. 2a) shows the schematic structure of an IgG antibody (left), which ischaracterized by a characteristic Y-shaped structure and is composed oftwo identical immunoglobulin (Ig) heavy and light chains, comprisingfour (V_(H)—C_(H)1-C_(H)2-C_(H)3) and two immunoglobulin domains(V_(L)—C_(L)), respectively. The V-domains are the highly variableantigen binding regions of IgH and IgL chains, whereas the C_(H) andC_(L) domains represent the constant region domains. The variable regiondomains of IgH chains are encoded by V, D and J gene segments, whereasthe variable region domains of IgL chains are encoded by only V and Jgene segments, which need to be assembled from germline immunoglobulingene loci (FIG. 2 b) and 2 c) during early B lymphopoiesis, by theprocess known as V(D)J recombination.

Antibody IgH and IgL chains are covalently held together by disulphidebridges, which couple the identical IgH chains together at a locationclose to the flexible hinge region, i.e. between the C_(H)1 and C_(H)2domains, whereas additional disulphide bridges between the C_(H)1 andC_(L) domains, as depicted, are covalently coupling IgH and IgL chains(FIG. 2 a left).

Fab fragments are univalent fragments of full-length antibodies onlycontaining V_(H)—C_(H)1/V_(L)—C_(L) domains coupled by a naturaldisulphide bridge, which can either be derived by enzymatic papaincleavage from full-length antibodies, or which can be expressed asrecombinant proteins by expressing C_(H)2-C_(H)3 deleted IgH chainstogether with IgL chains. Additional fragments of fully human antibodiesare single chain variable domain fragments (scFv-fragments), which onlycomprise the variable region domains of IgH and IgL chains that arecoupled by a synthetic linker or an artificial disulphide bridge. Theexpression of either full-length antibodies, or antibody fragments, asthe depicted Fab and scFv fragments, may also be expressed as bindingproteins in order to realize the invention.

FIG. 2 b) schematically depicts the process of V(D)J recombinationoccurring on a germline IgH chain allele, resulting in the assembly ofthe coding regions of antibody V_(H) domains. The variable domains ofIgH chains in vertebrate species are encoded by a multitude of V, D andJ gene segments, which are separated in germline configuration. DuringV(D)J recombination occurring during early lymphopoiesis, one selectedV, D and J gene segment is site-specifically rearranged to generate aunique coding region for an antibody V_(H) domain. V(D)J recombinationin the IgH chain locus is an ordered process and starts withrearrangement of a selected D to a selected J gene segment, usually onboth IgH chain alleles. Only after D to J gene rearrangement, oneselected V region is site-specifically joined to the already assembledDJ region, thereby generating a V-D-J ORF encoding the V_(H) domain. Theprocess of V(D)J recombination is dependent on the expression ofprecursor lymphocyte specific recombination activating genes (RAG) 1 and2.

FIG. 2 c) schematically depicts the process of V(D)J recombinationoccurring on a germline IgL chain allele, resulting in the assembly ofthe coding regions of antibody V_(L) domains. The variable domains ofIgL chains in vertebrate species are encoded only by V and J genesegments, which are separated in germline configuration, similar to thegene segments in the IgH chain locus. The generation of an antibodyV_(L) domains requires only one site-specific V(D)J recombination event,as depicted.

FIG. 3: This figure schematically illustrates the principle of stablegenetic modification of target cells for the expression of a bindingprotein of interest (BPOI) such as an antibody (alternatively labelled“X”) by retroviral transduction.

FIG. 3 a) depicts the schematic organization of a wild-type retroviralgenome (upper left), in which the genes for the structural andfunctional proteins Gag, Pol and Env are located in between so-called 5′and 3′ long-terminal repeat (LTR) sequences flanking the retroviralgenome. The 5′LTRs are important for the expression of the retroviralgenes and also for the replication of the retroviral genome in theinfected host cell. Another important region in the retroviral genome isthe ψ (Psi) packaging signal, which is required for the packaging of theretroviral RNA during replication and/or production of retroviralparticles.

For the generation of recombinant retroviral particles, the gag, pol andenv genes may be removed from a wild-type retroviral genome, so thatonly 5′ and 3′ LTRs and the ψ (Psi) packaging signal remains. For theconstruction of recombinant retroviral vectors it is then convenient tointroduce a multiple cloning site (MCS) containing several unique andconvenient restriction enzyme sites. This design, as depicted ontop/right, represents the simplest retroviral transfer vector.

For the expression of recombinant retroviruses allowing the expressionof a recombinant protein (e.g. a binding protein of interest (BPOI) “X”)such as an antibody, minimally an open reading frame (ORF) of a BPOIneeds to be inserted into an “empty” retroviral transfer vector, as the5′LTR region has a promoter activity able to drive expression of anydownstream positioned gene. However, in order to improve expressionlevels, expression of a gene of interest (e.g. a BPOI-“X”) mayoptionally be driven by an additional heterologous promoter (Prom.), andoptional addition of a marker gene, e.g. downstream of the 5′LTRpromoter and ψ packaging signal, as depicted here, may allow selectionand/or tracking of retrovirally transduced constructs.

FIG. 3 b) schematically illustrates the procedure of retroviraltransduction of target cells resulting in the stable expression of aBPOI-“X” such as an antibody. For this, first, a recombinant retroviralconstruct containing an expression cassette for a BPOI-“X” istransiently transfected into a retroviral packaging cell line (PCL),expressing structural and functional retroviral proteins Gag, Pol andEnv of a wild-type retrovirus (left). A retroviral PCL can be generatedby either stably or transiently transfecting expression constructs forthe Gag, Pol and Env proteins into a suitable and easy to transfect cellline (e.g. standard human 293 HEK cells, or mouse NIH 3T3 fibroblasts).Two to three days post transfection, the recombinant retroviral genomes,containing the BPOI-“X” gene are packaged into replication incompetentretroviral particles, which accumulate in the cell culture supernatantof the PCL. The retroviral particles are replication incompetent,because they lack the genes for the functional retroviral Gag, Pol andEnv proteins and therefore, they can deliver their genetic payload intoa target cell only once, a process that is called retroviraltransduction, or single round infection. During retroviral transductionthe packaged RNA of a recombinant retrovirus is introduced into thetarget cells, where it is reverse transcribed into cDNA, which is thenstably integrated into the target cell genome. Two to three days afterretroviral transduction, a gene of interest, like the BPOI-“X”, is thenpermanently expressed by the target cells, due to the integration of thecDNA retroviral construct into the host cell genome.

FIG. 4: This figure shows the schematic design of preferred types ofretroviral expression constructs that can be used to realize theinvention. The drawing depicts the schematic design of retroviralvectors contained in a standard DNA cloning plasmid backbone (closedblack line); the relevant genes and regions for the retroviral genomeare highlighted. One preferred vector generation, depicted in panel (a),whose detailed cloning is described in FIGS. 5 and 6, and provided inExample 1, contains the cDNA coding regions for human Igγ₁H chains andIgκL chains driven by a strong constitutive CMV promoter (Prom) andflanked up- and downstream by the Igκ intron enhancer (κiE) and 3′κenhancer (3′κE) elements, promoting somatic hypermutation to the Vcoding regions of the IgH and IgL chains. The retroviral IgH and IgLchain expression constructs additionally contain open reading frames forthe antibiotic resistance markers hygromycinB (hygro^(R)) and puromycin(puro^(R)), respectively, allowing the selection of stable integrationof the IgH and IgL chain constructs applying respective antibiotic drugselection to cultures of retrovirally transduced vertebrate cells. Inaddition, convenient, unique restriction enzyme sites are highlighted,allowing the straightforward replacement of V coding regions withHindIII and Eco47III, or the replacement of the entire IgH and IgL chaincoding regions by using the restriction enzymes HindIII and NotI. Thisway, from one existing IgH or IgL chain expression construct different Vregions and even entire collections of V regions can easily be clonedinto the disclosed expression vectors.

(b) In this panel, another class of preferred vectors is described,which carry a replacement of the variable coding region by a DNAfragment, in which the variable coding region is still separated into V,D and J gene segments (for the IgH construct) and V and J gene segments(for the IgL chain construct) in “quasi-germline” configuration. Whileotherwise identical to the retroviral expression vectors provided in (a)these V(D)J-recombination competent retroviral vectors first need toundergo site-specific rearrangement of the V, optionally D and J genesegments, in order to generate a coding region for a variable bindingdomain of a IgH or IgL chain. The detailed cloning of such a vectorallowing the expression of IgH chains after V(D)J recombination isdescribed in FIG. 11.

A unique feature of these constructs is their capability to generatediverse V domain coding regions in V(D)J recombination active cells insitu, e.g. in precursor lymphocytes expressing endogenous RAG1 and RAG2proteins. Because the process of V(D)J recombination is not precise, adiverse collection of variable coding region sequences may result fromone individual retroviral vector within a given set of V, D and J genesegments for IgH, or a given set of V and J gene segments for IgL. Thediversity in the joining of V, optionally D and J gene segments is dueto a combination of exonuclease activity, TdT mediated N-regionaddition, and P nucleotide generation, which may all contributeindividually or jointly to coding joint diversification. As the V, D andJ gene segments have been cloned in a fashion that different V, D and Jgene segment family members can be easily replaced by unique restrictionenzyme sites, a limited number of constructs generated and introducedinto V(D)J recombination competent host cells, can result in an enormousdiversity of in situ generated binding protein diversity. As thesevectors contain additional κiE and 3′κE elements, conferring somatichypermutation to an V(D)J-rearranged V domain coding region, a primaryin situ generated collection of diverse binding proteins can optionallyfurther be mutagenized by an AID-dependent somatic hypermutationprocess. This way, the entire process of generation of antibodydiversity in vivo, can be recapitulated in situ and in vitro using thedisclosed retroviral constructs and host cells exhibiting V(D)Jrecombination activity (e.g. precursor lymphocytes), and in which AIDmediated somatic hypermutation is active, or can be activated.

(c) This panel schematically depicts yet another design of retroviralconstructs that can be used to realize the invention. Here, theexpression of the IgH and IgL coding regions is driven by the 5′LTRpromoter of the retroviral backbone and the expression of IgH and IgLchains is coupled to the expression of GFP and YFP autofluorescencemarkers, respectively, allowing the tracking and isolation of IgH andIgL expressing cells simply by analyzing the transduced cells for greenand yellow fluorescence. These constructs are very useful forcontrolling the multiplicity of infection of “selector cells” withoutfurther labelling procedures.

A legend of symbols used in FIGS. 4 a) to c) for important DNA sequencesincluded in the construct is provided. The subdivision of the IgH andIgL coding regions into variable domains (V_(H) and V_(L)) allcontaining endogenous leader (L) sequences, hinge (H), constant (C_(H)1,C_(H)2, C_(H)3, C_(L)), and membrane-spanning coding regions (M1/2,because this region is encoded by two exons) is provided for a betterunderstanding of the illustrations.

FIG. 5 a-e illustrates the cloning strategy for the construction of aretroviral IgH (human Igγ₁ isotype) expression vector, disclosed indetail in Example 1, and in the basic design provided in FIG. 4( a). Thecloning of expression constructs for both membrane bound IgG as well assecreted IgG is depicted, as detailed in Example 1—unique restrictionenzyme sites in the plasmid maps are provided for general referencepurposes. Based on the final retroviral chain expression construct, asdisclosed in FIG. 5 e herein, any other V_(H) domain coding region, or acollection (library) of diverse V_(H) domain coding regions can beintroduced into the vectors using the unique HindIII and Eco47IIIrestriction enzyme sites, by replacing the existing V_(H) region withsaid any other V_(H) domain coding regions. FIG. 5 a depicts a firstpreparatory cloning step, in which an Eco47III restriction site(circled) is removed from the commercially available pLHCX vectorbackbone by site-directed mutagenesis, as described in Example 1. Thisgenerates the retroviral vector backbone pLHCXm1, in which the Eco47IIIrestriction enzyme site can later be re-introduced for the cloning andreplacement of V_(H) domain coding regions. The advantages of usingEco47III for this purpose is based on the fact that Eco47III is the onlyrestriction enzyme site that can be introduced directly at the borderbetween human V_(H) and Cγ₁ coding regions, without changing the aminoacid composition of expressed human Igγ₁H chains. FIG. 5 a furtherillustrates, how cloned fragments of the human γ₁ constant region genes,either with, or without membrane spanning exons M1/M2 are cloned intothe pLHCXm1 backbone using unique HindIII and ClaI restriction enzymesites present in the MCS of pLHCXm1. The fragments were designed tocontain additional flanking Eco47III and NotI restriction enzyme sitesfor later cloning purposes, as detailed in Example 1. FIG. 5 b) showsthe plasmids maps of the cloning intermediates without V_(H) domaincoding regions, and it is shown, how a particular V_(H) coding regionflanked by HindIII and Eco47III sites is cloned into the constructs.These constructs, which are thus generated, are depicted in FIG. 5 c,and would in principle be sufficient to confer the expression of humanIgγ₁H chains in any recipient cell line. However, the possibility toadditionally mutagenize V_(H) coding regions in an AID-dependent manner,is an aspect of this invention and two additional cloning steps aredisclosed, in which the core κiE element with additional flankingsequences is cloned into a unique BglII site, upstream of the CMVpromoter of the expression cassette (FIG. 5 c bottom and FIG. 5 d) andin which the 3′κE element with some flanking DNA sequence is cloned intothe unique ClaI site downstream of the expression cassette for humanIgγ₁H chains. This results in the final expression vector for eithermembrane bound or secreted human Igγ₁H chains, for which the plasmidmaps are provided in FIG. 5 e. These constructs correspond to theschematic plasmid maps that have already been disclosed in FIG. 4 a, buthere with precise restriction enzyme maps and drawn to scale.

FIG. 6 a-d illustrates the detailed cloning strategy provided in Example1, for the construction of retroviral IgL (human IgκL isotype)expression construct, whose basic design was already provided in FIG. 4(b). Based on the final retroviral IgκL chain expression construct, asdisclosed in FIG. 6 d herein, any other V_(L) domain coding region, or acollection (library) of diverse V_(L) domain coding regions can beintroduced into the vectors using the unique HindIII and Eco47111restriction enzyme sites, by replacing the existing V_(L) region withsaid any other V_(L) domain coding region(s). The cloning strategy forthe retroviral IgL chain expression vectors required preparatory cloningsteps, in order to generate a modified retroviral vector backbone, intowhich the desired elements could be cloned using convenient restrictionenzyme sites as depicted. In a first step, from commercial plasmid pLPCXan undesired Eco47III site was removed from the ψ (Psi) packaging signalby site-directed mutagenesis as described in Example 2, resulting inmodified plasmid pLPCXm1 (FIG. 6 a). In a second step a novel pLPCXm2backbone was generated by ligating a large, AscI-BlpI digested fragmentfrom commercial plasmid pLHCX with an AscI-NcoI fragment from pLPCXm1(FIG. 6 b). For both fragments the non-compatible BlpI and NcoI DNA endsneeded to be filled up with nucleotides using Klenow fragment asdescribed in Example 1. Into the resulting pLPCXm2 backbone the constantregion for a human κL chain (Cκ) has been inserted via HindIII and ClaIas shown (FIG. 6 b). Similar to the cloning strategy for human IgHchains, the human Cκ fragment was further flanked by Eco47III and NotIsites to facilitate additional cloning procedures. After insertion ofthe human Cκ fragment, one selected human Vκ element was cloned into theconstruct via unique HindIII and Eco47III sites (FIG. 6 c). Thisconstruct would in principle be sufficient to confer the expression ofhuman IgκL chains in any recipient cell line. However, like in the casefor the IgH chain expression constructs (FIG. 5 a-e), additional κiE and3′κE elements were cloned into the construct into the unique BglII andClaI sites upstream and downstream of the IgκL chain expressioncassette, identical to the cloning strategy of the IgH chain constructs(FIGS. 6 c and 6 d). In the final constructs also the Vκ domain codingregion can then be target for AID-mediated somatic hypermutation. Thefinal construct corresponds to the schematic plasmid map that isdetailed in FIG. 4( b), but here the precise restriction enzyme maps areincluded and drawn to scale.

FIG. 7: This figure illustrates the cloning strategy for a retroviralexpression construct for activation induced cytidine deaminase (AID). Asdepicted, the commercial pLPCX retroviral vector backbone was used and aspecific RT-PCR-fragment from mouse splenic cDNA containing the AIDcoding region was cloned into the unique XhoI restriction site of thepLPCX vector using compatible XhoI restriction enzyme sites insertedinto the PCR amplication primers, as described in Example 2.

FIG. 8: This FIG. 8 a and continued onto 8b) illustrates the detailedcloning strategy, also provided in Example 2, for a retroviral reporterconstructs with and without IgκL chain enhancer elements, allowing theidentification and quantitation of somatic mutations by reversion of adefined EGFP stop mutation.

FIG. 9: This figure provides an experimental proof-of-concept that thedisclosed retroviral vectors allow AID mediated somatic mutation ofsequences, like preferably antibody V coding regions, cloned downstreamof the V-promoter elements. Panel (a) shows an analysis of AIDexpression by Western-blotting of five selected FA-12 A-MuLV transformedcell clones that had been stably transfected with a retroviral AIDexpression construct, whose cloning was depicted in FIG. 7. TheWestern-blot analysis shows a distinct AID-specific signal of ca. 25 kDin FA-12 transfectant clones 1 through 4, but not in transfectant 5 andalso not in the non-transfected negative control (NC). Transfectant 3was used for further testing of retroviral reporter vectors forAID-mediated somatic hypermutation (SEM), which is depicted in panel(b): Here the retroviral reporter constructs of FIG. 8 (once with andonce without Igκ enhancer elements) were retrovirally transduced intoAID expressing and AID non-expressing FA-12 transfectants and 5,respectively. As expected, only when reporter constructs containing theenhancer elements were transduced into AID-expressing FA-12 transfectantclone 3, was it possible to detect green revertant transductants at a0.2% frequency 10 days post transduction. From these 0.2% green cells,100 individual cell clones were isolated by single cell sorting andthese clones were re-analyzed for green fluorescence by FACS afterexpansion. The vast majority of the single cell sorted clones (95%)displayed homogeneous green fluorescence expression at the samefluorescence intensity as the medium green fluorescence signal of the0.2% green cells originally sorted, and similar to the representativeGFP expression pattern provided at the lower left panel of FIG. 9 b,confirming that the original green population was due to reversions ofthe EGFP stop mutation. Four clones showed a bimodal green fluorescencepattern, as representatively depicted in the middle FACS histogram andonly 1 of the 100 single cell sorted cloned displayed hardly any greenfluorescence (right-hand FACS histogram).

FIG. 10: This figure illustrates the sequence of the EGFP coding regionwith an engineered stop mutation that was used to clone an EGFP reporterconstruct for quantitating somatic hypermutation. In panel (a) it isshown, which of the four nucleotides have been mutated in codon 107 and108 of the wildtype EGFP open reading frame (see nucleic acid sequenceggcaactacaagacccgc (SEQ ID NO:34) which encodes amino acid sequenceGNYKTR (SEQ ID NO:36)) thereby generating a stop-codon in codon 107 andgenerating a lysine to threonine amino acid change in codon 108 (seenucleic acid sequence ggcaactagtatacccgc (nucleotides 313-330 of SEQ IDNO:36). These four nucleotide changes additionally resulted in theintroduction of unique SpeI restriction enzyme site, as indicated thatcould be used as a diagnostic marker for stop codon reversions uponsomatic hypermutation. The G-nucleotide of the TAG stop codon isembedded in a so-called RGYW sequence motif, which is known to be ahotspot for somatic hypermutation. In 24 revertant clones analyzed bySpeI restriction enzyme digestion, it could be confirmed that the sitewas rendered resistant to SpeI digestion (and hence was mutated). In tenof these clones sequence analysis revealed that the G nucleotide in theoriginal TAG stop-codon had been mutated to a C nucleotide, resulting ina TAC codon, thereby confirming the restriction enzyme analysis, anddemonstrating that AID-mediated somatic mutation had been targeted tothe G in the RGYW motif.

(b) This panel shows the entire ORF of the mutated EGFP that was clonedinto the retroviral Igγ1H chain construct already disclosed in FIG. 5(e), instead of a V_(H) domain coding region. The nucleic acid codingsequence, the N-terminal amino acid sequence, and the C-terminal aminoacid sequence of the mutated EGFP are set forth in SEQ ID NO: 36, 37 and38, respectively.

FIG. 11( a) and (b): Illustration of the detailed cloning strategy of aV(D)J recombination competent retroviral IgH chain expression vector, asdisclosed in detail in Example 4.

FIG. 12: Proof-of-concept that retroviral constructs requiring V(D)Jrecombination of V, D and J gene segments in “quasi-germline”configuration can give rise to productively rearranged heavy chainexpression constructs and Ig+ cells upon transduction into RAG1/RAG2positive precursor lymphocytes. Panel (a) contain data showing thegeneration of surface immunoglobulin positive cells (0.04%, upper rightquadrant of left FACS plot) after transduction of a V(D)J recombinationcompetent retroviral expression vector (detailed description of cloning,see FIG. 11) into A-MuLV transformed preB cell line 230-238. Theimmunoglobulin expression is coupled to EGFP expression using constructsas schematically illustrated in FIG. 4 c. Therefore, immunoglobulinexpressing cells can only be generated in the population of green (i.e.stably transduced) cells. The right staining panel shows re-analysis ofsurface immunoglobulin expression after a single round of FACSenrichment and expansion of the rare (0.04%) surface immunoglobulincells for 8 days in tissue culture. After this one round of enrichment,the combined frequency of immunoglobulin positive cells had increased to17.8% (as expected detectable in the green, i.e. the stably transducedpopulation) from which PCR amplicons have been obtained and sequenced.(b). As a representative example, this panel shows a DNA sequence (clone225, with amino acid translation on top) obtained from a PCR ampliconderived from surface immunoglobulin cells after one round of enrichmentthat had been transduced with “quasi-germline” V(D)J recombinationcompetent retroviral vectors. As a reference, the sequences of thecoding regions of the V, D and J gene segments (SEQ ID NO: 39, 41, and42 respectively) are provided in (b) at the top, also with amino acidtranslation on top of the V and J gene segments (SEQ ID NO: 40 and 43respectively), as the D segment sequence can be read in three differentreading frames, depending on the junctional diversity after V(D)Jrecombination. Intervening sequences between the V, D and J genesegments in “quasi-germline” configuration are depicted with dots. Thesequence of recovered clone 225 clearly represents a bona fide V(D)Jrearrangement event, with typical features of nucleotide loss and TdTcatalyzed N-sequence additions clearly detectable at the coding jointsbetween the assembled V, D and J gene segments (all interveningsequences had been lost from clone 225). The sequence of clone 225exhibited an open reading frame and, apart from the aforementionedvariations at the coding junctions, did not contain additional somaticmutations in the V, D and J sequences Amino acid sequences YYCAKDQ andWELDAFDIW are set forth in SEQ ID NO: 45 and 47 respectively. Nucleicacid sequences tattactgtgcgaaagatcaa and tgggagcttgatgattttgatatctgg areset forth in SEQ ID NO: 44 and 46 respectively.

FIG. 13: Data showing the testing of a panel of different A-MuLVtransformed murine preB cell lines for the susceptibility to ecotropicMLV-derived vector gene transfer. 1×10⁵ cells were transduced with a MOIof 0.5 using a vector preparation having packaged the reporter gene EGFPencompassing transfer vector LEGFP-N1. Transduction was carried out asdetailed in Example 5. Two days post transduction, gene transfer wasdetected by expression of EGFP using FACS. Except for preB cell line18/81, all other tested A-MuLV transformed preB cell lines weresusceptible for transduction at frequencies ranging between 40-60% underthe applied conditions, and can, in principle, be used for the currentinvention. Untreated naïve target cells served as negative controls andshowed no green fluorescence (not shown).

FIG. 14: Characterization of a panel of murine preB cell lines forintracellular expression of endogenous IgM heavy chains (cy-μH), inorder to identify cells devoid of endogenous murine antibody expressionthat can be used as selector cells for retrocyte display. Cells werepermeabilised and stained using anti-murine IgM heavy chain antibodiescoupled to FITC (FL1). Untreated cells served as negative controls. Theexperiment shows that cell lines FA-12, 1624-5, 1624-6, 18/81-c18-11,and 40E1 had practically undetectable endogenous antibody expression,and can thus be used in a method of the present invention.

FIG. 15: Illustration of the complexity of retroviral expression vectorsfollowing the design disclosed in FIG. 4( c) and the experimentalprinciple for the generation of a IgH and IgL chain shuffled antibodylibrary. (a) The retroviral vector libraries IgH(650)-LIB-IRES-GFP andIgL(245)-LIB-IRES-YFP encompass defined collections of coding regionsfor heavy (HC) and light chains (LC) for fully human antibodies with acomplexity of 650 and 245 different, fully sequenced clones,respectively. Both vectors harbour the packaging sequence Psi (ψ),flanking long terminal repeats (LTR) and an internal ribosome entrysignal (IRES). Parallel to the expression of an antibody polypeptidechain mediated by the viral promoter in the 5′LTR, the IRES enables thecoupled expression of the reporter gene gfp and yfp, respectively. Uponviral gene transfer into selector cells, this allows for the convenientdetection and enrichment of successfully transduced and immunoglobulinchain expressing cells using FACS.

(b) Generation of a collection of fully human antibodies in transformedpreB cells. In order to generate transient packaging cells, libraries ofretroviral transfer vector libraries encoding heavy chains of humanantibodies (IgH(650)-LIB-IRES-GFP) are co-transfected with a packagingconstruct (pVPack-GP) and an envelope construct (pVPack-Eco) intosuitable recipient cells. Two days post transfection, the generatedvector particles library having packaged the respective transfer vectorlibrary are harvested and employed to transduce selector pre B cells.Transduced cells expressing the transferred heavy chains and thereporter gene gfp are expanded enriched using FACS. Following expansion,cells are subjected to a second transduction. This time, theIgL(245)-LIB-IRES-YFP library is transferred followed by expansion andenrichment of cells expressing YFP and human light chains employingFACS. The resultant population constitutes a fully human antibodydisplaying a defined human antibody library expressed by 1624-5 cells,containing a complexity of maximally 159′250 clones.

FIG. 16: This figure shows how a two-step transduction with IgH-IRES-GFPand IgL-IRES-YFP libraries has been performed at conditions ensuring atransduction, resulting in clonal expression of polypeptide chains inthe vast majority of the transduced cells. 1.5×10⁶ 1624-5 murine A-MulVtransformed preB cells were suspended in 1 ml of tissue culture mediumsupplemented with different quantities of vector particle supernatant(diluted 1:1; 1:5; 1:20; 1:50; 1:100; 1:200) containing recombinantretroviral vectors encoding IgH and IgL chain libraries IgH-LIB-IRES-GFPor IgL-LIB-IRES-YFP, respectively, already described in FIG. 15. Toensure that the majority of the transduced cells received single copiesof transfer vectors integrated into the host cell genome, cellsdisplaying gene transfer efficiencies lower than 10% (MOI<0.1, asdetected by expression of the coupled GFP or YFP reporters) wereenriched using FACS sorting four days post infection. Cells wereexpanded for six days and subjected to a second transduction employingvector particles having packaged the light chain coding regions ofantibodies at a dilution of 1:5 as described above. Here, GFP-positivecells selected for heavy chain expression were infected with vectorparticles transducing the IgL-LIB-IRES-YFP library and vice versa. Fourdays post infection, transduced cells expressing GFP and YFP wereenriched using FACS. Approximately 20% of the cells showed GFP and YFPexpression after the second transduction. To secure that only singlevector integrations occurred per cell about one third of the populationswere enriched that revealed only low or moderate expression of thereporter gene transduced in the second round (approximately 8%).

FIG. 17: Titration of IL-15 staining with a population of preB cellsexpressing an anti-IL-15 reference antibody by FACS, in order to defineoptimal conditions allowing optimal IL-15 antigen staining conditionsfor Retrocyte Display experiments. The staining procedure, as disclosedin detail in Example 7, included a titration of the IL-15 antigen in therange of 2.5 μg/ml-0.1 μg/ml, at two different concentrations of apolyclonal, biotinylated anti-IL-15 secondary antibody, as indicated,which was detected with streptavidin-PE conjugate by FACS. Surface Ig+cells were counterstained with an anti-IgκL chain-APC antibody. As canbe seen, optimal IL-15 staining is accomplished at a concentration of0.1 or 0.5 μg/ml IL-15 antigen, and using 3 μg/ml of the secondary,polyclonal anti-IL-15 antibody.

FIG. 18: Analysis of FACS-identification of an anti-IL-15 referenceantibody expressing preB cell line (PC=positive control), which wasspiked into a diverse library of antibody expressing preB cells atdifferent dilutions, by using the optimized IL-15 staining conditionsillustrated and determined in FIG. 17. The top-left panel shows the IgκLchain-APC/IL-15 double staining of control preB cells transduced with acombination of IgH and IgL chain libraries, whose generation was alreadyshown in FIG. 16 (NC=negative control). The top right panel shows theIgκL chain-APC/IL-15 double staining of preB cells transduced withretroviral expression vectors encoding IgH and IgL chains of a referenceIL-15 antibody (PC=positive control), as disclosed in detail in Example7. The FACS profile of the NC cells shows that approximately 50% of theAb-library transduced cells are surface-Ig+, as detected by theanti-IgκL chain-APC staining. However, none of the surface-Ig+ cellsdisplays binding to IL-15. In contrast, the PC cells, in which more than90% of the cells expressed surface Ig a specific IL-15-antigen bindingis apparent by a specific signal on the x-axis. As expected, the higherthe expression of surface-Ig on the PC cells, the more pronounced theshift for the specific IL-15 signal, resulting in a diagonal stainingpattern of surface-Ig+/IL-15 binding cells, which is highlighted by aelipse-shaped gate, as indicated. The panel on the bottom, showingdouble-FACS stainings for surface-Ig and IL-15-binding in five differentdilutions of PC cells spiked into the NC random antibody libraryexpressing cell population shows that a specific anti-IL-15 referenceantibody expressing PC cells can be detected at frequencies close to thepercentage of PC cells spiked into the NC cell library.

FIG. 19: Proof of concept for the enrichment of a IL-15-reactive cellpopulation by Retrocyte Display from a diverse antibody library, asdisclosed in detail in Example 7. The top panel shows FACS stainings forGFP/YFP expression (y-axis), indicative of the frequency ofIg-retrovector transduced cells, and IL-15/anti-IL-15-bio α-axis),indicative of specific IL-15 staining. The top-left panel shows thetwo-colour FACS analysis of untransduced control preB cells (NC=negativecontrol), the top-middle panel shows the two-colour FACS analysis ofpreB cells transduced with an anti-IL-15 reference antibody as apositive control (PC). The top-right panel shows the same two-colourFACS staining of a population of cells that have been transduced with asingle IgH chain encoding retroviral vector encoding the IgH chain ofthe reference anti-IL15 antibody in combination with a diverse, >7×10⁴different IgκL chain library. This IgL chain shuffled library thereforecontains potentially>7×10⁴ different antibodies, and expectedly, even byvery narrow gating for antibody-expressing and IL-15 reactive cells, asindicated in the top-right FACS-profile, very few IL-15 reactive cellscould be detected (here 2.42%, due to the gating close to the negativepopulation, as indicated). The enriched population was expanded intissue culture, and the identical staining procedure and FACS-sortingwas repeated three times, as shown for the three FACS stainings underidentical conditions in the lower three FACS panels. As can be seen,consecutive enrichment/cell expansion cycles resulted in a population ofcells that was almost 100% positive for antibody expression and evenmore positive for IL-15-reactivity than the original PC cell line. Thisdata shows clearly that by repeated FACS sorting and expansion a highlyantigen-reactive cell population can be successfully enriched to anessentially 100% antigen-reactive cell population from almostundetectable antigen-reactive cell populations using three consecutiverounds of Retrocyte Display.

FIG. 20: This figure illustrates and confirms the specific IL-antigenreactivity of 4 representatives of 24 individual cell clones establishedafter single cell sorting from a 3 times IL-15 antigen enriched cellpopulation, as described in FIG. 19. The 4 selected cell clones aredesignated clone F, H, V and W, and all show specific IL-15 reactivityon GFP/YFP positive cells, indicative of the stably transduced, Igencoding retroviral vectors. As expected, higher GFP/YFP expressingcells, expressing higher antibody levels showed higher IL-15specificity, leading to characteristic diagonal staining signals in theIg/IL-15 double stainings. All cell clones showed specific IL-15reactivity, as demonstrated by omission of the IL-15 antigen in thestainings, which led to a loss of IL-15-specific reactivity (not shown).The data provide proof of concept that Retrocyte Display is an efficientmethod to obtain antigen-reactive cell clones at high frequencies fromcell populations initially showing almost undetectable antigen-reactivecells.

FIG. 21: This figure provides a second proof of concept for successfulRetrocyte Display enrichments of antigen-reactive cells by illustratingthe successful enrichment of IL-1β antigen-reactive cells to anessentially 40% antigen-reactive cell population using three consecutiverounds of retrocyte display cell enrichment/tissue culture expansion,starting from a minimally IL-1beta-reactive cell population in theinitial cell population. Double stainings for GFP/YFP expression(indicative of antibody expression) and IL-1β reactivity are provided.FACS stainings on top are provided for non-transduced preB selectorcells (as negative control ═NC, top-left) and cells co-transduced withretroviral vectors encoding an anti human IL-1β specific referenceantibody SK48-E26 (as positive control=PC, top right), as indicated. Thebottom panels show the FACS stainings for antibody expression and IL-1βreactivity of an antibody library generated by shuffling of a diverseIgL chain library of >1.2×10⁵ individual IgL chain clones against theIgH chain of the SK48-E26 reference antibody, before (0× enriched) andafter 1, 2 and 3 Retrocyte Display enrichment rounds, as indicated andas disclosed in detail in Example 8. These data provide an independentproof of concept using a second antigen that Retrocyte Displayexpression and enrichment is a powerful means to enrich a population ofantigen-specific cells from initially almost undetectable levels.

FIG. 22: This figure shows confirmation of IL-1β antigen reactivity of anovel antibody identified by Retrocyte Display, as disclosed in detailin Example 8. From the 3× enriched cell population, shown in FIG. 21, 24individual cell clones have been established by single cell sorting.From these 24 cell clones, 12 clones harboured a novel IgL chain, termedLCB24, as disclosed in Example 8. The IL-1β specificity of the novelLCB24 IgκL chain co-expressed with the IgH chain of the IL-1β specificreference antibody SK48-E26 (see Example 8) was analyzed by FACS uponre-transduction of the cloned and sequence characterized IgL and IgHchain retroviral expression vectors into the original selector cellline. The FACS stainings show analysis of antibody expression (viaGFP/YFP) and IL-1β reactivity by two colour FACS, as indicated. Asexpected no IL-1β reactivity is detected in non-transduced selectorcells (NC=negative control, left), whereas a clear IL-1β specificstaining is detected in positive control cells expressing IgH and IgLchains of reference antibody SK48-E26 (middle). A similar, IL-1βspecific, signal is detectable in antibody expressing selector cellstransduced with the SK48-E26 reference antibody IgH chain vector and thenovel, fully human LCB24 IgκL chain, cloned from IL-1β specificRetrocyte Display cell clones (right).

FIG. 23: Confirmation of lack of cross-reactivity to IL-15 of novelantibody encoded by LCB24 IgkL chain/SK48-E25 IgH chain. The two leftFACS stainings show negative and positive controls for the IL-15 FACSstaining assay, as indicated (NC=negative control, untransduced selectorcells, PC=positive control, selector cells transduced with IgH and IgLchain vectors encoding an anti-IL-15 reference antibody). The two rightFACS stainings show no IL-15 reactivity on antibody expressing cellseither encoding the novel antibody composed of SK48-E26 IgH and LCB24IgL chain, or on cells expressing the original SK48-E26 IgH/IgLcombination. This demonstrates that the novel antibody composed ofSK48-E26 IgH and LCB24 IgL chain is not only specific for IL-1β, butthat it is not generally cross-reactive (or sticky) to other proteins,like IL-15.

FIG. 24: This figure illustrates the successful enrichment ofstreptavidin-APC-Cy7 antigen-reactive cells by three consecutive roundsof Retrocyte Display cell enrichment/tissue culture expansion, from anantibody library generated by shuffling of a diverse IgL with a diverseIgH chain library as disclosed in Example 9. Streptavidin-APC-Cy7reactive cells were enriched by three consecutive rounds of high-speedcell sorting, followed by cell culture expansion, as indicated. Thebinding-specificity of antibody expressing cells for thestreptavidin-APC-Cy7 antigen is demonstrated by analyzing FACS profilesof the sequentially enriched cell populations in the presence (lowerpanel) and absence (top panel) of the antigen. This demonstrates a proofof concept for the efficient enrichment of antigen-specific by RetrocyteDisplay in the absence of any reference antibody that could be used forchain shuffling approaches.

FIG. 25: The data presented in this figure provide evidence for thespecificity of the 3 times Retrocyte Display enriched cell populationdisclosed in FIG. 24 for specific reactivity to the Cy7 fluorochrome ofthe strepatavidin-APC-Cy7 tandem dye. For this, non-transduced selectorcells, unenriched cells expressing an IgH/IgL chain library combinationand a 3 times strepatavidin-APC-Cy7 enriched cell population wereanalyzed by FACS for antibody expression (indicated by GFP/YFPfluorescence) and reactivity to different streptavidin-fluorochromeconjugates, as indicated. The 3 times streptavidin-APC-Cy7 enriched cellpopulation only bound to streptavidin-APC-Cy7, but not tostreptavidin-APC or streptavidin-APC-Cy5.5, and non-specific staining ofstrepatavidin-APC-Cy7 was also not detectable to either the selectorcells or the selector cells expressing a diverse antibody library. Thisprovides proof of concept for the efficient and highly specificRetrocyte Display enrichment of specific antibodies from cellsexpressing a diverse antibody library, without the need of antibody IgHor IgL chains from antigen-specific reference antibodies.

FIG. 26: Two novel human antibodies identified by Retrocyte Display,sharing the same IgH chain show specific binding to antigenstreptavidin-APC-Cy7. As disclosed in Example 9, two different IgH chainsequences (HC49 and HC58) and two different IgL chain sequences (LC4 andLC10) could be identified from single-sorted cell clones after threerounds of Retrocyte Display enrichment. In this figure, all possiblepairings of IgL chains LC4 and LC10 with HC49 and HC58 were examined forreactivity to the target antigen streptavidin-APC-Cy7. For this,combinations of retroviral expression vectors encoding the different IgHand IgL chains were transduced into selector cells as indicated and asdisclosed in Example 9. As illustrated, novel antibodies HC58/LC4 andHC58/LC10, both sharing the same IgH chain, displayed specific bindingto the streptavidin-APC-Cy7 antigen, whereas antibodies encoded byHC49/LC4 and HC49/LC10 did not show significant binding activity. Thespecific binding of the two novel antibody clones to the antigenstreptavidin-APC-Cy7 upon re-transduction into selector cells providesconclusive evidence that it is possible to use Retrocyte Display asdisclosed herein for the identification of rare antibody binders incomplex antibody libraries.

TERMINOLOGY

It is convenient to point out here that “and/or” where used herein is tobe taken as specific disclosure of each of the two specified features orcomponents with or without the other. For example “A and/or B” is to betaken as specific disclosure of each of (i) A, (ii) B and (iii) A and B,just as if each is set out individually herein.

Affinity maturation: A highly regulated immunological process ofantigen-driven improvement of the binding specificities of antibodiesproduced by antigen-stimulated B lymphocytes, mostly occurring ingerminal centers. The process is caused by somatic hypermutation largelytargeted to the coding regions for the variable domains of antibodiescoupled with the selective expansion and survival of B lymphocytesgenerating higher affinity antibodies.

Antibody: This term describes an immunoglobulin whether natural orpartly or wholly synthetically produced. The term also covers anypolypeptide or protein comprising an antibody antigen-binding site, likeheavy chain only antibodies from for example camels or lamas. Afull-length antibody comprises two identical heavy (H) chains and twoidentical light (L) chains. In its monomeric form, two IgH and two IgLchains assemble into a symmetric Y shaped disulphide linked antibodymolecule that has two binding domains formed by the combination of thevariable regions of IgH and IgL chains.

Antibodies can be isolated or obtained by purification from naturalsources, or else obtained by genetic engineering, recombinant expressionor by chemical synthesis, and they can then contain amino acids notencoded by germline immunoglobulin genes. A fully human antibodycomprises human heavy and light chains i.e. variable and constantdomains from the human species. A chimeric antibody comprises variableregion domains from one vertebrate species combined with constant regiondomains of another vertebrate species. The constant domains of achimeric antibody are usually derived from a human antibody orantibodies. Humanised antibodies can be produced by grafting CDRs ofnon-human antibodies onto framework regions of IgH and IgL variabledomains of human origin.

Antibody fragment: It has been shown that fragments of a whole antibodycan perform the function of binding antigens. Examples of bindingfragments are (i) the Fab fragment consisting of VL, VH, CL and CH1domains; (ii) the Fd fragment consisting of the VH and CH1 domains;(iii) the Fv fragment consisting of the VL and VH domains of a singleantibody; (iv) the dAb fragment, which consists of a VH or a VL domain;(v) isolated CDR regions; (vi) F(ab′)2 fragments, a bivalent fragmentcomprising two linked Fab fragments; (vii) single chain Fv molecules(scFv), wherein a V_(H) domain and a V_(L) domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site; (viii) bispecific single chain Fv dimmers; and(ix) “diabodies”, multivalent or multispecific fragments constructed bygene fusion. Fv, scFv or diabody molecules may be stabilized by theincorporation of disulphide bridges linking the V_(H) and V_(L) domains.Minibodies comprising an scFv joined to a CH3 domain may also be made.Other examples of binding fragments are Fab′, which differs from Fabfragments by the addition of a few residues at the carboxyl terminus ofthe heavy chain CH1 domain, including one or more cysteines from theantibody hinge region, and Fab′-SH, which is a Fab′ fragment in whichthe cysteine residue(s) of the constant domains bear a free thiol group.In some cases a heavy or a light chain may also be considered to be anantibody fragment. As a skilled person will readily appreciate, all theabove antibody fragments display at least one function of the wholenative antibody from which said fragments are derived and are thustermed ‘functional’ fragments.

Antigen: Any biomolecule or chemical entity that can be bound by thevariable domains of immunoglobulins (or antibodies).

Binding protein: This term defines one protein of a pair of moleculesthat bind one another. The binding partner of a binding protein isusually referred to as a ligand. The proteins of a binding pair may benaturally derived or wholly or partially synthetically produced. Oneprotein of the pair of molecules has an area on its surface, or acavity, which binds to and is therefore complementary to a particularspatial and polar organization of the other protein of the pair ofmolecules. Examples of types of binding pairs are antigen-antibody,biotin-avidin, hormone-hormone receptor, receptor-ligand,enzyme-substrate. The present invention is preferably concerned withantigen-antibody type reactions.

Complementary determining region (CDR): This term refers to thehypervariable regions of the heavy and light chains of animmunoglobulin. CDRs are the regions in the three dimensional structureof an immunoglobulin that directly establish contact to antigen. Anantibody typically contains 3 heavy chain CDRs and 3 light chain CDRs.The CDRs are usually the most diverse parts of antigen receptors.

Domain: A structural moiety of a biomolecule that is characterized by aparticular three dimensional structure (e.g. variable or constant regiondomains of immunoglobulins that are structurally related, such asIg-like domains that can be found in many molecules of the immunesystem, which belong to the so-called Ig-superfamily).

Germinal center: A distinct histological structure in peripherallymphoid organs (e.g. lymph nodes or spleen) where cognate interactionsbetween antigen presenting cells and between different lymphocytepopulations occur, resulting in the proliferative expansion ofantigen-reactive lymphocytes, as well as affinity maturation and classswitch recombination of antibodies produced by antigen reactive Blymphocytes.

Germline configuration: The unrearranged configuration of genes and geneloci, as they are inherited from the parents, and as they will be passedon to further generations through the germline. DNA recombination eventsoccurring in somatic cells, like e.g. V(D)J recombination inlymphocytes, lead to the reshuffling or loss of genetic information oncertain gene loci and therefore to a change of the genes from thegermline configuration.

PreB lymphocyte: A precursor B lymphocyte is characterized by theexpression of particular precursor B cell specific genes, like e.g. theλ5 and V_(preB1) and V_(preB2) genes, and the expression of precursorlymphoid specific factors involved in V(D)J recombination (e.g. RAG-1,RAG-2). In addition, precursor B lymphocytes are characterized by thepresence of DJ_(H) on both heavy chain alleles or at least oneV_(H)DJ_(H) rearrangement on at least one immunoglobulin heavy chainallele, while the light chain gene loci are still in unrearranged,germline configuration, such that the preB cells cannot express completeantibodies.

Primary lymphoid organs: Organs, in which lymphocytes develop fromhematopoietic stem cells, in mice and humans e.g. the bone marrow, thethymus, and during fetal life, the liver.

“Quasi-germline” configuration: The artificial arrangement of V,optionally D, and J gene segments with flanking recombination signalsequences cloned from germline immunoglobulin gene loci into artificialgenetic constructs, such that the arrangement of the V, optionally D,and J gene segments in such artificial genetic constructs still allowssite-specific recombination of the gene segments into a variable codingregion by the process of V(D)J recombination.

Somatic mutation: A process in somatic cells resulting in theintroduction of point mutations into specific regions of the genome.When this occurs at a high frequency (>10⁻⁴ mutations per basepair percell division) it is known as somatic hypermutation.

V(D)J recombination: This is the process for generating antibody andT-cell receptor diversity and is the method by which functional antibodygenes are created. It involves the rearrangement of many gene segmentsthat code for the heavy and light chain proteins of immunoglobulins, andit only occurs in lymphocytes.

Transfecting/Transfection: In the context of eukaryotic cells this isthe process of introducing nucleic acid sequences into eukaryotic cells,usually associated with using chemical and/or physical methods.

Transforming/Transformation: In the context of eukaryotic cells this isthe process of immortalizing a cell for the establishment of acontinuously proliferating cell line.

Transducing: The process of delivering DNA into vertebrate cells via theproduction of recombinant viruses. For this, a packaging cell line,expressing structural proteins for viral particles is transfected with arecombinant viral DNA construct comprising the regulatory elements forpackaging of the viral DNA construct into the viral structural proteins.By this, recombinant viruses are produced that can be used to infect(mammalian) target cells leading to the introduction of the geneticinformation cloned into the recombinant viral genome.

Vector/Construct: An artificially generated nucleic acid sequence whichcan be used to shuttle nucleic acid elements between different organismsand species, and which can further be used to propagate, amplify andmaintain genomic information.

DETAILED DESCRIPTION

Antibodies, or immunoglobulins, are the most widespread class of bindingproteins that have proven to be particular useful for therapeutic andfor diagnostic applications. Therapeutic antibodies have developed intothe commercially most successful class of biologic drugs and there iscontinued interest in novel and powerful methods to developantibody-based therapeutics (Baker, 2005).

Antibodies consist of two identical heavy (H) chain and light (L) chainglycoproteins that are covalently linked via disulphide bonds (FIG. 2a). Each immunoglobulin heavy (IgH) and light chain (IgL) polypeptidecomprises an N-terminal variable domain that varies between differentantibodies and a C-terminal constant region, that is identical betweendifferent antibodies belonging to the same immunoglobulin subtype(isotype) (FIG. 2 a). The combination of IgH and IgL chain variabledomains creates the antigen binding pocket of an antibody and determinesits specificity, whereas the constant regions determines the immuneeffector function of an antibody. The variability of immunoglobulins intheir variable domains results from the fact that V_(H) and V_(L)domains are encoded by a multitude of gene segments, that are designatedV (variable), D (diversity), and J (joining) gene segments. During thedifferentiation of B lymphocytes one V, one D (only present in the IgHchain locus) and one J gene segment is randomly selected in each celland is site-specifically rearranged in order to generate the codingregion for V_(H) or V_(L) domains. This site specific geneticrecombination process only occurs in precursor lymphocytes and is knownas V(D)J recombination (Grawunder et al., 1998) (also refer to FIGS. 2 band 2 c). The rearrangement of gene segments is mediated byrecombination-activating gene (RAG) 1 and 2 products. Due to themultitude of V, D, and J gene segments, and imprecision in gene segmentjoining, an enormous repertoire of different V region specificities canbe generated by the millions of B lymphocytes produced by the immunesystem every day (Grawunder et al., 1998). Because the immunoglobulinheavy chain gene locus contain V, D and J gene segments, the codingregion for a V_(H) domain of an antibody requires two sequential V(D)Jrearrangement events, whereas the immunoglobulin gene locus lacks D genesegments and the V_(L) coding region is generated by one V toJ-rearrangement event (FIG. 2 c). Therefore, the junctional diversitythat is generated by V(D)J recombination in the CDR3 region of IgHchains is greater than the CDR3 junctional diversity that is generatedby only one rearrangement event for the IgL chains. In addition, at theearly B cell differentiation stages, during which IgH chain generearrangements occur, the enzyme terminal deoxynucleo-tidyltransferase(TdT) is expressed, that is able to add non-templated nucleotides at theD to J and V to D junctions (so-called N-sequence diversity),additionally diversifying the IgH chain CDR3 repertoire. In contrast,the CDR3 repertoire of the IgL chain, which is formed upon V to J genesegment joining later during B cell differentiation, when TdT expressionis largely downregulated (Li et al., 1993), is somewhat less complex.Apart from the generation of CDR3 diversity in V_(H) and V_(L) codingregions, both IgH and IgL chain repertoires can further be diversifiedby the process of somatic hypermutation that is triggered in mature Bcells during the course of a T cell dependent immune reaction(Papavasiliou & Schatz, 2002). The somatic mutations are specificallytargeted to the V_(H) and V_(L) coding regions, and are mediated by theB lineage specific enzyme activation-induced cytidine deaminase(abbreviated AID, see: Papavasiliou & Schatz, 2002). As a consequence ofsomatic hypermutation occurring during immunization, cells expressinghigher affinity antibody mutants against the immunogen are positivelyselected in the course of an immunization mostly occurring in germinalcenters, and resulting in an enrichment of cells producing higheraffinity antibodies. These antibodies now also accumulate mutations inCDRs 1 and 2, a process, which is referred to as affinity maturation ofthe antibody repertoire. The AID-mediated diversification and specifictargeting to the V_(H) and V_(L) coding regions is significantlyincreased by the presence of cis-regulatory genetic elements or motifs,in particular enhancer elements of the IgH and IgL chain gene locus,located in the proximity of the rearranged V_(H) and V_(L) codingregions (Bachl & Olsson, 1999).

Besides classical, full-length therapeutic antibodies, additionalformats of binding proteins, comprising fragments of fully humanantibodies, e.g. so-called F_(ab) fragments (FIG. 2 a), single-chainF_(v) fragments (FIG. 2 a), nanobodies, only consisting of single V_(H)domains, etc., are also increasingly being explored as therapeutic anddiagnostic agents. However, it is clear to a person skilled in the art,that such functional antibody fragments can easily be derived fromfull-length antibodies by either standard biochemistry methods onprotein basis, or by conventional molecular biology methods, if thecoding information of a desired full-length antibody is available.

Traditionally, monoclonal antibodies directed against a molecule orepitope of interest (antigen) are generated by immunization of smalllaboratory, or farm animals, e.g. mice, rats, rabbits, or goats anddonkeys, respectively. After repeated immunizations, animals are eitherbled for the isolation of polyclonal antibodies from their blood serumor, for the generation of monoclonal antibodies, the animals aresacrificed in order to isolate lymphocytes from secondary lymphoidorgans, like lymph nodes or the spleen. Isolated lymphocytes are fusedto immortal myeloma cells for the generation of hybridomas, which aresubsequently subcloned and screened for the secretion of monoclonalantibodies exhibiting desired functional properties, like e.g. bindingto a particular antigen or target.

From the first breakthrough in antibody engineering, namely thedevelopment of hybridoma technology for the generation of so-calledmonoclonal antibodies by Köhler and Milstein (Köhler & Milstein, 1975),it took a long time until monoclonal antibodies could be used for thetreatment of human disease.

The main reasons for the slow entry of antibodies into the clinic wereinitial setbacks associated with using rodent antibodies for treatinghuman patients. If such antibodies are infused into the immune system ofa patient, the immune system recognizes the rodent antibodies as aforeign protein and mounts an immune response against these antibodies,including the generation of neutralizing antibodies (known asHAMA=human-anti-mouse antibody response). A HAMA response can lead to asignificant decrease in the half-life and, hence, the efficacy of theapplied antibody, and can even lead to severe side-effects, if theimmune system overreacts against the injected non-human protein.

Therefore, it was of great medical and commercial interest to developtherapeutic antibodies that were “more” similar to human antibodies.Initially, this was achieved by means of genetic engineering of existingrodent antibodies, resulting in the development of either chimeric orhumanized antibodies (Clark, 2000). Chimeric antibodies are generated byfusing the variable binding domains of a rodent antibody to the constantregions of a human antibody, using standard genetic engineering andcloning techniques. Humanized antibodies, in contrast, are generated byonly transferring the complementarity determining regions (CDRs) of avariable domain from a rodent antibody to the variable region frameworkof a human antibody, which is also done by standard molecular biologytechniques. While the procedure for generating chimeric antibodies isstraightforward, these antibodies still contain 33% xenogeneic sequencesand harbour a significant potential for immunogenicity (Clark, 2000). Infact, immune responses to the mouse parts of a chimeric antibody arewell documented and are referred to as HACA-responses (HACA=humananti-chimeric antibody).

In contrast to the aforementioned, the immunogenic potential ofhumanized antibodies is further decreased. However, the procedure ofgenetically engineering humanized antibodies and at the same timemaintaining original binding affinities and specificities of the rodentantibodies after CDR grafting is not trivial, and often requiresextensive additional optimization by repeated mutagenesis and screeningcycles. For the above-mentioned reasons, chimerization and humanizationapproaches have become less appreciated in recent years as a method ofchoice for the development of therapeutic antibodies.

The development away from chimeric and humanized antibodies for thedevelopment of therapeutic antibodies, has also been driven by thedevelopment of innovative technology platforms allowing the developmentof “fully human” antibodies, which by amino acid sequence are identicalto human serum antibodies. Fully human antibodies theoretically arethought to cause the least immunogenicity and side-effects in humanpatients.

The two most established “fully human antibody” development platformsare:

A) Human immunoglobulin transgenic mouse technology, in which largetransgenes of germline human immunoglobulin heavy and light chain geneloci have been introduced into the mouse genome (Green & Jakobovits,1998; Jakobovits et al., WO98/24893 A2). In order to use thesetransgenic mice for the development of human antibodies, thesetransgenic mouse strains have been crossed to gene knock out mousestrains harbouring functional deletions in their endogenous mouseimmunoglobulin heavy and κ light chain gene loci. Thus, these humanimmunoglobulin transgenic mice mount a largely human humoral immuneresponse upon immunization, with the exception that approximately halfof the antibody producing cells still harbour endogenous mouse λ lightchains, which are therefore useless for further therapeutic antibodydevelopment, and need to be discarded.B) Phage display technology, which is based on the expression (display)of highly diverse libraries of antibody fragments (e.g. as single chainF_(v) or F_(ab) fragments) on the surface of bacteriophages of E. coli(Clackson et al., 1991; McCafferty et al., WO 92/01047 A1). For theidentification of specific binders, phage libraries of appropriatecomplexity, quality and origin are bound to immobilized antigens(“panning”), in order to enrich for phage clones binding to theimmobilized antigen. After several rounds of panning, sequences ofselected binding clones are determined. A variation of the method is thecompletely cell-free ribosome display technology, in which antibodyfragments are not displayed on phage, but rather expressed by in vitrotranscription and translation, under conditions, where the translatedbinders still “stick” to ribosomes (Hanes & Plückthun, 1997). For eitherphage or ribosome display one crucial step is the re-engineering ofbinding fragments into full-length antibodies, which are then expressedin vertebrate cells. After re-engineering of phage selected clones intoa full-length antibody format and vertebrate cell expression it needs tobe analyzed, whether the antibodies can be expressed adequately andwhether the original phage binding characteristics are still maintained,which may not necessarily be the case.

Although the human immunoglobulin transgenic mouse and the phage displaytechnologies have had a major impact on therapeutic antibodydevelopment, both technology platforms have advantages and disadvantagesassociated with them.

One advantage of the transgenic mouse technology is that it can deliverhigh affinity antibodies, due to the natural affinity maturationoccurring in these mice upon in vivo immunization, and it has beendemonstrated that the affinity profile of human antibodies towards agiven antigen derived from human transgenic mice can be comparable tothat of wild-type mice. However, several disadvantages associated withthe human immunoglobulin transgenic animals are:

1) If the transgenic animals are tolerant to the antigen, most often dueto high structural similarity to endogenously expressed host proteins,the generation of high affinity antibodies against such “conserved”antigens can turn out to be very difficult, or even impossible. 2) Likein normal wild-type animals, antibodies from human immunoglobulintransgenic animals are preferentially generated against strong antigenicepitopes, which can make it a challenging task to develop an antibodyagainst functional, but weak epitopes of therapeutic value. 3) Lastly,human immunoglobulin transgenic animals cannot be used for affinityoptimization of existing antibodies. The reason for this is that thetime frames required for the generation of transgenic animals, just forthe optimization of one given antibody clone, are too long. Such anapproach would involve the generation of two IgH chain and IgL chaintransgenic mouse strains for one particular antibody and would thenadditionally require the genetic backcrossing of these two transgenicstrains to at least two knock-out animal strains deficient for bothendogenous immunoglobulin heavy and for κ light chain expression, aprocess that would require several breeding generations and extendedtime frames.

Similar limitations as described above, apply to a recently describedtechnology-platform based on the development of mice, in which thegermline variable (V), diversity (D) and joining (J) gene segments ofthe mouse immunoglobulin heavy and light chain gene loci have beenreplaced by (parts of) human germline V, D and J gene regions bysite-specific gene targeting (Murphy & Yancopoulos, WO 02/066630 A1). Inthese “immunoglobulin gene-knock-in” mice, the murine V, D and J genesegments have been site specifically replaced by those of the humanimmunoglobulin gene heavy and light chain gene loci via homologousrecombination in the mouse germline. In contrast to human immunoglobulintransgenic mice, which produce fully human antibodies, this mouse straintherefore produces “reverse-chimeric” antibodies, carrying human antigenbinding regions on a mouse constant region backbone.

Phage and/or ribosome display approaches have the perceived advantage ofbeing very fast technology platforms, because the identification offirst binders from complex libraries of binding proteins can beaccomplished within a few weeks. However, phage display is alsoassociated with significant disadvantages. 1) Due to the absence of anyaffinity maturation in the system, it is not trivial to identify highaffinity binders from a phage or ribosome display screen. In order toaddress this problem, extremely complex phage libraries representingmore than 10¹² clones have been developed. But even using such complexlibraries, initial binding clones often have suboptimal affinity to theantigen and such binders usually still need to be optimized usingadditional tedious and time consuming optimization procedures. 2) Inphage display, only antibody fragments, such as scF, or F_(ab) fragmentsare expressed, because the phage genome can only accommodate codingregions for relatively small-sized molecules. 3) Binding proteins haveto be fused to carrier proteins such as the phage gIII protein. Theresultant fusion proteins frequently reveal lower antigen-reactivitycompared to their parental antibodies or binding-active proteins(Hoogenboom & Chames, 2000). 4) Phage display does not easily allow fora controlled assembly of proteins that attain a binding phenotypethrough the formation of homo- and hetero-multimer formation, becausee.g. dimeric proteins are forced to assemble by covalent linkermolecules. However, in the case of antibody engineering a properlyregulated assembly of immunoglobulin heavy and light chains isessential, as not every antibody heavy chain is able to pair with anylight chain. 5) Bacteria- or bacterial phage-based systems do notprovide appropriate posttranslational modifications (glycosylation,myristoylation and the like) of the displayed protein of interest, whichoften negatively influences the binding characteristics of the expressedproteins. 6) Prokaryotic expression results in different protein foldingof proteins in comparison to vertebrate cells, as the cytoplasmaticenvironment dramatically differs from eukaryotic or vertebrate hostcells, e.g. In redox potential and the lack of chaperones. 7) Phagedisplay systems are subsequently subjected to antigen binding- orcapture-assays to enrich reactive cells under quite non-physiological“panning” conditions, which may lead to the identification of a largepercentage of false-positive binders that eventually need to bediscarded.

As a result of the abovementioned drawbacks, many phage-display selectedantibody fragments have mediocre affinity and/or may carry structuralartefacts. In addition, once phage display selected binders arere-engineered and expressed as full-length antibodies in vertebratecells, it may happen that phage selected antibodies are either poorly ornot at all expressible, or that they exhibit altered bindingcharacteristics.

In order to address some of the limitations of human immunoglobulintransgenic/knock-in mouse technology and phage display, an alternativetechnology has recently been developed, which involves the geneticmodification of primary murine preB cells in vitro, resulting in cellsexpressing human antibodies, followed by their engraftment intoimmunodeficient recipient mice lacking a functional B cell compartment(Grawunder & Melchers, WO 03/068819 A1). This leads to a partialreconstitution of B cell subsets expressing human antibodies in theengrafted mice, which may subsequently be immunized with any desiredantigen or ligand. This technology can be used to either develop novelantibodies or binding proteins, or to optimize existing antibodies withregard to their affinity against a defined target (Grawunder & Melchers,WO 03/068819 A1).

The use of retroviral expression systems in this method is preferred,because gene transfer of single copies of expression constructs can betransferred into individual preB cells (Kitamura et al., 1995; Stitz Jet al., 2005), and also, because there is complete freedom of choice, asto which antibody expression constructs are being used for engraftmentfor the mice (e.g. antigen pre-selected antibody libraries, antibodylibraries from diseased patients, or individual antibody clones).Therefore, a particular advantage of this technology is its flexibilitythat it can be applied to de novo development of antibodies, as well asto the optimization of existing therapeutic antibody candidates.

Other rodent-based systems for the development of fully human antibodieshave been described, which involve the transplantation of humanhematopoietic progenitor cells isolated from human donors intoimmunodeficient mice (Mosier & Wilson, WO 89/12823 A1). In such humancell engrafted mice, human B cells may develop to some extent, however,despite recent improvements of this method (Traggiai et al., 2004), asatisfactory humoral immune response involving affinity maturation ofhuman antibodies is not achieved in such “humanized mice”. In addition,like in the case of human immunoglobulin transgenic or “knock-in” mice,existing antibodies cannot be optimized.

Any kind of mouse based antibody technology platform usually requires invivo immunization which remains a time-consuming process when comparedto in vitro approaches.

Therefore, in addition to the aforementioned mouse-related approaches, avariety of alternative in vitro technologies have recently beendeveloped. However, it still needs to be proven how efficient thesesystems will be in developing high quality, high affinity antibodyproducts. One in vitro system is based on the isolation ofantigen-enriched memory B cells from human patients with a particulardisease that can be isolated and then be immortalized by Epstein-BarrVirus (EBV) transformation in vitro, followed by the screening forantigen-reactive EBV lines (Lanzavecchia, WO 04/76677 A2). Similar inconcept, but different in methodology are approaches in which antibodyproducing plasma cells from patients with an acute disease status arefirst isolated from peripheral blood and then immortalized by fusion tonon-producing heteromyelomas, followed by their screening for desiredantibody producers (Lang et al, WO 90/13660 A2). Alternatively, methodshave been described aiming at the isolation of B cells from vaccinatedor immunized individuals followed by isolation and cloning of specificantibody genes either from cell populations (Lawson & Lightwood, WO04/106377 A1; Schrader, WO 92/02551 A1) or by single-cell PCR (Muraguchiet al., WO 04/051266 A1). However all these technologies rely on theavailability of relevant B cell populations in human patients and arequite limited in their general application and are therefore mainly usedfor the identification of anti-infective therapeutic antibodycandidates. Furthermore, neither affinity maturation, norantigen-directed development of antibodies, nor optimization of existingantibodies is possible with any of the human B cell-based screeningapproaches.

Therefore, additional alternative in vitro methods have recently beendescribed involving the expression and screening of recombinantantibodies in eukaryotic cells using transient expression systems(Zauderer & Smith, WO 02/102855 A2 and Beerli et al, WO 08/055,795 A1).While these systems circumvent some of the bottlenecks of transgenicmice, phage display and human B cell derived technologies, these systemsstill are characterized by a number of limitations. First, the knowneukaryotic cell-based antibody expression/screening technologies do notconfer a stable expression pattern for recombinant antibodies,precluding repetitive enrichment cycles of antibody-expressing cellswith desired binding specificity. Second, none of the known approachesinvolving eukaryotic cell based antibody expression allows control forclonal expression of binder clones, which in the case of therapeuticantibody development makes it a challenging task to identify a matchingIgH chain and IgL chain pair with desired antigen or ligand bindingactivity. Third, the technology described in Zauderer and Smith (WO02/102855 A2) does not allow any in vitro mutagenesis, or geneticrecombination of the expressed antibodies, the method is a merescreening procedure. Therefore, aspects of affinity maturation ofbinding proteins are not addressed by this technique. Lastly, none ofthe eukaryotic expression/screening systems are compatible with the insitu generation of diverse antibody repertoires from individual antibodyexpression constructs exploiting the mechanism of V(D)J recombination ofimmunoglobulin heavy and light chain V, D and J gene segments.

An alternative method for the identification of biologically activepeptides and nucleic acids has been proposed by Jensen et al (EP 1 041143 A). The preferred method described in EP 1 041 143 A comprises aninitial screening procedure in which a large number of retroviralvectors can be introduced into cells such that the individual cell canexpress a number of different RNAs or peptides. The cells that show aphenotypic change are subsequently isolated and the retroviral DNA inthat clone can be isolated by PCR. This PCR product can then be used tore-transfect viral packaging cells to create further retroviral vectors.These retroviral vectors can then be used for the transduction ofdifferent cells and finally after a second cloning procedure the activesubstance can be identified. Essentially this method results in anindirect change in the phenotype of a cell by the importation ofbiologically active peptides or nucleic acids. This is in contrast tothe method of the present invention whereby the retrovirally transducedconstructs directly encode the binding proteins, preferably antibodies,to which the screening is directed. It should also be noted that thepeptides and nucleic acids described in EP 1 041 143 A differ greatly insize to the antibodies or antibody fragments identified by methods ofthe present invention.

A further method for retrovirus-based genomic screening is set out in WO03/083075 A2 (Bremel et al). This method relates to the expression andscreening of genomic DNA sequences encoding uncharacterised genes andproteins. A process is described in which a cell line is transduced witha retroviral expression construct such that a genomic DNA virus isinserted into the genome of the cell line as a provirus, and then theexpression of polypeptides from the provirus is analysed directly. Sucha method does not provide the opportunity for enrichment of the cellline, nor for the isolation and identification of the expressedpolypeptides before analysis is performed, which would detract from thehigh-throughput screening technology developed by Bremel and co-workers.

A recently published patent application (WO 08/055,795 A1) from Beerliand co-workers describes a screening platform for the isolation of humanantibodies, which utilises a Sindbis virus expression system. Anessential feature of this platform is the generation of starting librarywhere B cells specific for an antigen of interest are directly isolatedfrom peripheral blood mononuclear cells (PBMCs) of human donors.Recombinant, antigen-reactive scFv libraries are generated from thispool of B cells and screened by mammalian cell surface display by usinga Sindbis virus expression system. Similar to phage display, one of thedrawbacks to this system is that the scFvs of interest need to bere-engineered and expressed as full-length IgGs in vertebrate cells.Such a process can be associated with a loss in affinity of the antibodyof interest on conversion since these antibodies may not express well invertebrate cells and/or may exhibit altered binding characteristics.

In contrast, the invention disclosed herein comprises a unique extremelypowerful combination of methods for the development and optimization ofbinding proteins, preferably antibodies, or fragments thereof. Incomparison to mouse-based technologies, the main advantages of theinvention disclosed herein are complete flexibility in terms ofoptimization and de novo development of antibodies, and the speed toidentify specific binders in short periods of time. As all aspects ofthe invention are realized in vitro, there is no limitation with regardto the development of antibodies against antigens, which are highlyconserved across species, or that may be toxic in experimental animals.

In comparison to phage display based technologies, the key advantages ofthe invention disclosed herein is that the binding proteins, inparticular antibodies, can be expressed as full-length antibodies, in avertebrate cell, and preferably in a B lymphocyte environment, i.e. thenatural host cell of antibodies, ensuring most natural and properprotein folding, correct posttranslational modification, and a qualitycontrol for heavy and light chain pairing.

In comparison to human B cell approaches the key advantages of thedisclosed invention are the complete flexibility with regard todevelopment of antibodies against any desired target, the possibility toaffinity optimize existing antibodies, a complete freedom of choice asto which type of antibody is expressed in the system (antigen-enriched,synthetic, from patients, under conditions of IgH and IgL chainshuffling, etc.).

In comparison to other eukaryotic cell-based expression systemsinvolving either plasmid based expression constructs or non-integratingviral vectors, key advantages of the disclosed invention of ‘RetrocyteDisplay’ are that stable, sustained and clonal expression can beachieved by use of retroviral gene transfer technology. The stable,sustained and clonal expression of recombinant antibodies in the targetcells allows repetitive enrichment cycles of antigen- or ligand specificcells, including the possibility to isolate and to expand monoclonalcells for identification of the antibody genes. Moreover, the inventiondisclosed herein additionally allows the additional generation ofgenetic diversity upon retroviral transduction into vertebrate hostcells in situ using single retroviral constructs by either exploitingthe lymphocyte specific mechanism of V(D)J recombination, or exploitingthe process of somatic hypermutation for further mutagenesis of bindingproteins.

Therefore, in comparison to any of the known technologies for thedevelopment of therapeutic antibodies known in the art, the method ofretrocyte display disclosed herein provides unique, novel and powerfulsolutions for many evident limitations that pre-existing technologiessuffer from.

The invention disclosed herein is broadly applicable to the expression,screening and identification of binding proteins specifically binding toa ligand or antigen of interest. While the invention can be performedwith any binding protein, including but not limited to monomeric, homo-or hetero-multimeric membrane bound receptors, like T cell receptors,cytokine, or chemokine receptors, but also with other scaffold proteins,the preferred binding proteins according to the invention arefull-length antibodies, with fully human antibodies being particularlypreferred. However, it is to be understood, that any (functional)fragment of an antibody, including, but not limited to single chain Fvfragments (scF_(v)), Fab fragments, F(ab′)2, single V_(H) or V_(L)domains, single heavy or light chains or any combination thereof, withany naturally occurring or artificially engineered modification may beused to realize the invention. With regard to full-length antibodies,the invention is particularly applicable to any kind of artificiallyengineered or designed modifications of antibody binding regions, e.g.those generated by site-, or region-directed mutagenesis, fusion ofnaturally occurring sequences from different antibodies, randomizationof CDR sequences, DNA shuffling, error-prone PCR, just to name a fewmethods by way of illustration.

A preferred method for the expression of binding proteins according tothe invention is to use retroviral vector-mediated transduction ofvertebrate host cells.

The use of retrovirus vectors has been investigated for many years inthe field of gene therapy. For example, to engineer adeno-associatedvirus (AAV) vectors that can be targeted to specific cell types, Peraboet al., (WO 03/054197 A2) have inserted randomised sequences encodingtargeting peptides into the viral capsid gene, at a site critical forbinding to the primary cellular receptor, and produced AAV librariesthat displayed the peptides in the context of the viral capsid. Theselective pressure provided by the culture environment drove theselection by means of the ability of the viral clones to accomplishevery step in the infection process, namely binding, uptake, uncoating,nuclear translocation, replication, and gene expression. By using thistechnique, vectors were generated that efficiently transduced leukemiacells. Whilst such a technique may be useful to generate viral mutantsthat infect target cells previously resistant to infection by wild-typeAAV, it does not provide for the generation of diverse collections ofbinding proteins in vitro.

As such, the methods described in the present application for theexpression of binding proteins have several key advantages over anyother methods known in the art for the expression of recombinantproteins in eukaryotic and/or vertebrate host cells.

1) Recombinant retroviral constructs stably integrate into the host cellgenome and thereby confer a stable and sustained expression phenotype ofthe binding protein. 2) By utilization of appropriate ratios ofretroviral particles to target cells, termed “multiplicity of infection”(MOI), preferably performed at a MOI of equal or less than 0.1, theretroviral transduction can be controlled, such that the majority ofretrovirally transduced cells are genetically modified by only onerecombinant retroviral construct integrating into the host cell genomeresulting in clonal expression of an at least one desired bindingprotein. Because clonal expression of binding proteins greatlyfacilitates the identification and cloning of individual bindingproteins, this aspect therefore represents a preferred embodiment of theinvention. However, in an alternative embodiment, the invention may alsobe realized using retroviral transduction at MOTs of greater than 0.1.

Despite the aforementioned advantages of retroviral transduction as abasis for retrocyte display, expression of recombinant binding proteinsin vertebrate host cells may also be achieved by alternative methods,like, for instance, but not limited to, transient or stable DNAtransfection, RNA transfection, or by transfer of DNA-based viralvectors, like adeno-viral or poxvirus-based vectors—albeit none of theaforementioned alternative methods allows for an easily controllablestable and clonal expression of binding proteins in vertebrate hostcells.

Preferred vertebrate host cells for the realization of the invention arecells of the B lymphocyte lineage, in particular precursor Blymphocytes, which often lack endogenous antibody expression, but whichexpress favourable accessory proteins, like e.g. chaperones for properprotein folding and antibody assembly, or accessory membrane proteinsfacilitating membrane deposition of antibody molecules, like e.g. the Bcell specific Igα or Igβ proteins.

The principle of expression of recombinant proteins by retroviraltransduction in vertebrate host cells is an established procedure andinvolves the construction of recombinant retroviral vectors, that arerelatively small (maximal size of recombinant DNA to be incorporated:8-10 kB) and that can be cloned and manipulated by standard molecularbiology methods as plasmid vectors, from which the retroviral RNA genomecan be transcribed. A wild-type retroviral genome only contains threegenes, gag, poi and env, which encode the nuclear core proteins, aretroviral integrase, protease, RNAse, and a reverse transcriptase, andenvelope proteins, respectively (FIG. 3 a). In addition, the retroviralgenome contains cis-regulatory sequences, like the Psi (ψ) sequencerequired for packaging of the retroviral RNA genome into virusparticles, a polyA signal for retroviral transcript termination, andlastly, so-called 5′- and 3′-long-terminal repeats (LTRs) containingpromoter elements and signals for retroviral integration into the hostcell genome (FIG. 3 a). For the construction of recombinantretroviruses, the gag, pol and env coding regions of a wild-typeretrovirus are replaced by any expression cassette for a gene ofinterest (FIG. 3 a), including relevant cis-regulatory elements, likepromoters or enhancers. In order to stably integrate such recombinantretroviral genomes into a host genome, a plasmid vector containing aretroviral genome needs to be transiently or stably transfected into aso-called retroviral packaging cell line (PCL), expressing the viralstructural proteins encoded by gag, pol and env in trans in a transientor stable fashion, and therefore allowing the packaging of therecombinant viral genome (the transfer vector) into replicationincompetent retroviral particles (FIG. 3 b). These retroviral particlesallow for a single-round infection (transduction) of target cells (FIG.3 b). The entry of the retroviral particle into target cells is mediatedby a specific interaction of the Env protein with a specific receptor onthe target cell. Thus, the nature of the Env protein determines thetropism of the retroviral particles to specific host cells expressingthe cognate receptor. Ecotropic retroviruses are restricted to rodentcells, amphotropic retroviruses may infect various species includingrodent and human cells and pantropic retroviruses may infect anyreplicating cell with a cell membrane, as the cell entry occurs viastructures present on all eukaryotic cell membranes. Retroviral vectorparticles with a variety of different tropisms can also be generatedusing heterologous envelope proteins of other viruses such as gibbon apeleukemia virus (GaLV), vesicular stomatitis virus (VSV) or HIV and SIVor even cellular membrane proteins, just to name a few by way ofillustration—a technique known as “pseudotyping”. Following cell entry,a retrovirus can deliver the viral genome into the host cell, where theviral proteins mediate reverse transcription of the genome into cDNA andeventually its stable integration into the host cell genome, allowingstable expression of the delivered genes (FIG. 3 b). In a preferredembodiment of the invention ecotropic MLV particles are used to mediategene transfer into murine B cells. However, it will be appreciated byany person skilled in the art that any infectious retroviral vectorpseudotyped with any other envelope or transmembrane protein can beemployed to realize the invention, provided that it mediatestransduction in any appropriate target selector cell independent fromtheir parental donor species, cell type or their expression of a cognatereceptor mediating vector cell entry.

To achieve retroviral vector-mediated gene transfer, vector-containingretroviral particles (containing transcripts of recombinant retroviralgenomes, or transfer vectors) can be harvested from the cell culturesupernatant of packaging cells either stably or transiently expressingtransfer vectors (FIG. 3 b). This can be carried out in a broad range ofprotocols and variations thereof, known to a person skilled in the art.Preferred embodiments of this invention include: 1) preparation ofcell-free retroviral particle containing supernatants using eitherpassage through an appropriate filter or a centrifugation step thatseparates packaging cells from vector particles. These retroviralparticle preparations are subsequently used to transduce vertebrate hostcells by co-incubation for a variable time frame or by performing aso-called “spin infection”. Here, a target cell suspension is mixed withretroviral particle containing medium and is subjected to low-speedcentrifugation (FIG. 3 b). 2) Alternatively, co-cultivation of targetcells with packaging cells enabling cell-to-cell contact or separationof both cell populations by a membrane which allows the passage ofretroviral particles but not of the packaging cells, can be performed toenable transduction of target cells.

As host target cells for retroviral transduction a preferred embodimentof the method is to use B-lymphocyte lineage cells from rodents that donot express endogenous murine immunoglobulin proteins, and that can betransduced with retroviruses of ecotropic host range. Cells of the Blymphocyte lineage have the advantage that they already express B cellspecific Igα and Igβ proteins that are favourable for cell surfaceexpression and anchoring of membrane bound, full-length immunoglobulins.In that regard, immunoglobulin negative plasma cell-derived cells, likee.g. myeloma cells, as for instance, but not limited to Sp2/0, NSO, X63and Ag8653 generally lack the accessory Igα and Igβ proteins formembrane immunoglobulin deposition. In such cases and in any othervertebrate host cell, in which Igα and Igβ proteins are not expressed,the method may still be applied, if expression of the Igα and Igβproteins is conferred upon transfection or transduction of expressionvectors for Igα and Igβ, a standard procedure for any person skilled inthe art. Thus, upon ectopic expression of both Igo and Igβ proteins, themethod may be realized with any vertebrate host cell line, provided thatretroviral particles with appropriate tropism are produced, that areable to transduce said vertebrate host cell line. In order to clarify,the innovation disclosed herein could be realized with any vertebratehost cell, if pantropic retroviral particles (for instance, but notlimited to particles pseudotyped with the G protein of VSV) are used inconnection with a host cell that has been modified to ectopicallyexpress the immunoglobulin anchor molecules Igα and Igβ.

Preferred cells of the B-lymphocyte lineage are for instance, but notlimited to precursor B-, B-leukemia or B-lymphoma cells, from anyvertebrate species, but also primary precursor B cells that can be grownin tissue culture for long periods of time. Precursor B lymphocyteswould represent ideal host cells for retroviral expression ofimmunoglobulins, as the majority of such cell lines, do not expressendogenous immunoglobulin proteins. In particular, as murine preB celllines can easily be obtained from any mouse strain by transformationwith Abelson-murine leukemia virus (A-MuLV). However, either primary,long-term proliferating preB cells, as well as A-MuLV transformed preBcells express the preB cell specific proteins VpreB and λ5, whichtogether form the so-called surrogate light chain, which, in the absenceof conventional light chains, can form preB cell receptor complexes ofimmunoglobulin heavy and surrogate light chain. Because it is desired toexpress immunoglobulins composed of recombinant heavy and light chains,preB cells are preferred that lack expression of surrogate light chaincomponents, comprising the gene products of the λ5, or VpreB1, or VpreB2genes either as single, double or triple-gene knockouts. As it is knownthat surrogate light chain can bind to heterologous heavy chains it isexpected that surrogate light chain expression may interfere at avarying degree with the screening of IgH/IgL pairs, but due to generallylow expression levels of surrogate light chain proteins in preB cells,the method may yet be realized using wild-type preB cells, expressingsurrogate light chain components. In summary, any vertebrate cell lineexpressing Igα and Igβ, and not expressing endogenous immunoglobulinproteins may be used as target host cells for the method, with surrogatelight chain deficient preB cells being the preferred host cells forrealizing the invention.

The preferred binding proteins to be expressed, screened and identifiedare full-length antibodies, and by amino acid sequence, fully humanimmunoglobulins. However, it shall be understood, that any bindingprotein capable of cell expression in vertebrate cells may be subjectedto screening and selection for specific ligand or antigen bindingaccording to the disclosed method. For instance, such binding proteinsmay include fragments of antibodies from any vertebrate species, likee.g. single chain Fv, Fab fragments (FIG. 2 a) or single V_(H) or V_(L)domains, or a heavy or light chain, preferably expressed in a way thatdeposition on the cell surface membrane is enabled. This could beachieved e.g. by fusion to membrane anchors of other type-Itransmembrane proteins, utilization of GPI-anchor domains or othermethods know in the art. Furthermore, the method would also beapplicable to other membrane bound proteins, e.g., but not limited to,monomeric or multimeric cytokine receptors, or dimeric T cell receptorsand like. Retroviral expression of immunoglobulin heavy and light chainsis preferably achieved by sequential transduction of separate retroviralexpression constructs for heavy and light chains. However, the inventioncan also be realized by performing a co-transduction of the targetcells, in which separate retroviral constructs for IgH and IgL chainsare being used. The separate expression of IgH and IgL chains fromdifferent retroviral vectors offers the advantage, that collections ofretroviral vectors encoding a diverse collection of immunoglobulin heavychains can randomly be combined with collections of retroviralexpression vectors encoding a diverse collection of immunoglobulin lightchains. This so-called heavy and light chain shuffling can create alarge degree of diversity of different immunoglobulin bindingspecificities, even when the total number of heavy and light chaincollections are limited (e.g. 10⁴ different heavy chains, randomlycombined with 10⁴ different light chains theoretically results in 10⁸different antibody specificities). Shuffling of collections of IgH andIgL chain vectors is preferably performed with a one sided shuffling,meaning that one polypeptide chain of an antibody is a single constructencoding a single antibody chain.

However, it is to be understood, that retroviral IgH and IgL chainexpression can also be achieved, if both proteins are encoded on thesame retroviral backbone (see below). In its easiest configuration theheavy and light chain expression is conferred by cloning of heavy andlight chain cDNAs into an empty retroviral vector, where expression isdriven by the promoter activity of the 5′LTR and proper RNA processingis mediated by the 3′LTR sequences (FIG. 3 a). The heavy chainconstructs should preferably contain their endogenous membrane-spanningcoding region, in order to allow optimal membrane deposition of therecombinant immunoglobulins. However, for those skilled in the art, itis obvious that also membrane spanning domains of other transmembraneproteins may be fused to the constant regions of antibodies, in order toassure surface deposition of the expressed modified immunoglobulins. Inparticular in the context of expression of antibody fragments, or theexpression of non-immunoglobulin binding proteins, differenttransmembrane regions of membrane bound proteins may be advantageous forcell surface expression of the binders.

Whereas cell surface expression of antibodies or fragments thereof is apreferred embodiment of the current invention, these biomolecules mayalternatively also be expressed as soluble, secreted proteins, such thatdetection of the antibody is performed in fluid phase. Such a form ofexpression could be advantageous, if the screening of single producerclones and binders involves an assay requiring soluble antibodies, or ifthe assay is performed in semi-solid media with an assay allowing thequantitation of expression levels and binding specificities on singlecell clones. Expression vectors for recombinant immunoglobulins may beemployed coding for all known immunoglobulin heavy and light chainisotypes, which in the case of fully human antibodies, allows theexpression of IgM, IgD, IgG₁, IgG₂, IgG₃, IgG₄, IgA₂ and IgE antibodies,either containing Igκ or Igλ light chains. In all retroviral expressionvectors for human heavy and light chains, it is preferred that only thevariable coding region of a human heavy and light chain shall bereplaced using unique restriction enzymes, like e.g., but not limited toHindIII and Eco47III, as depicted in the schematic drawing of retroviralantibody expression vectors (4a and 4b). This will allow easy cloningand replacement of variable coding regions in retroviral expressionvectors, either with V-region libraries or individual V-region codingregions, in-frame to the constant coding regions for immunoglobulinheavy and light chains. Such a scheme of only exchanging variable regiondomains, either aiming at generating expression vectors encoding asingle specificity, or aiming at generating a collection of bindingproteins will be favourable. In this regard full-length antibodies maybe expressed that contain variable region domains and constant regiondomains derived from different species (chimeric antibodies).

The simplest retroviral expression vector for binding proteins could beconstructed by insertion of a cDNA coding region for the binding proteinor gene of interest into an “empty” retroviral expression vectorbackbone (FIG. 3 a). Even in the absence of any selection marker and/orscreening marker (e.g. enhanced green fluorescent protein, EGFP)allowing the direct detection of transduced cells, the invention couldbe realized, because cells stably expressing binding proteins from theretroviral vectors can be identified and isolated based on the stableexpression of the binding proteins, either in secreted or inmembrane-bound form. However, various features included in theretroviral expression vectors are preferred. The first one is a strongconstitutive or an inducible promoter element driving the expression ofthe recombinant binding proteins, which are placed directly upstream ofthe coding cDNA regions (FIG. 4 a, b). Such promoters may be, forinstance, but not limited to, constitutive promoters, as the immediateearly CMV promoter, β-actin promoter, EF-1α promoter, or induciblepromoters, like tetracycline- or any other antibiotic-induciblepromoter, that may either upregulate or downregulate expression byaddition or removal of tetracycline or other antibiotics and derivativesthereof, like doxycycline. The inclusion of inducible promoter elementsin the retroviral expression constructs is another preferred embodiment,because it is known that in some retroviral vector backbones either5′LTR promoters or even strong constitutive promoters can be silenced.

In addition to promoter elements, it is a preferred embodiment toinclude marker genes in the retroviral expression constructs, whichsubsequently allow the selection and/or monitoring of stable retroviraltransduction of host cells without detection of the recombinant bindingproteins (FIGS. 4 a, b). Selection and/or screening markers areparticularly useful for the preferred two-step retroviral transductionprotocol, involving the sequential transduction of immunoglobulin heavyand light chain retroviral expression vectors. In a two-steptransduction protocol a vertebrate host cell is first transduced with atleast one retroviral expression construct encoding a firstimmunoglobulin polypeptide chain or chains, and after the first at leastone polypeptide chain is stably expressed, a second transduction with atleast one retroviral expression construct encoding the correspondingother immunoglobulin polypeptide chain or chains, then allowinggeneration of a complete antibody or collection of antibodies. If aselection or screening marker is used for the selection or screening fora successful first transduction event, it is very useful to optimize theco-transduction frequencies of at least two retroviral expressionconstructs encoding separate chains of a multimeric binding protein,like antibodies. The use of selection and/or screening markers istherefore strongly preferred.

Selection markers, conferring resistance to antibiotics useful for theselection of mammalian cells, include, but are not limited to, e.g.genes for puromycin, neomycin, hygromcin B, mycophenolic acid,histidinol, bleomycin, and phleomycin resistance. For the expression ofmultimeric proteins, like antibodies, encoded by separate retroviralconstructs, it is preferable that expression of different polypeptidechains are linked to different selection markers, thereby allowingseparate selection for the stable transduction of correspondingexpression constructs.

Marker genes, allowing monitoring of retroviral transduction into hostcells include, but are not limited to genes, conferringauto-fluorescence to transduced cells, like e.g., but not limited togreen fluorescent protein (GFP), enhanced green fluorescent protein(EGFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP)and red fluorescent protein (RFP). Alternatively, cell surface markerscould be used such as CD7 or truncated variants thereof, CD34 ortruncated variants thereof, or low affinity nerve growth factorreceptor. In a preferred embodiment the expression of these antibioticselection markers, fluorescence markers or cell surface markers iscoupled to the expression of the recombinant binding protein viaso-called internal ribosomal entry sequences (IRES), which in vertebratecells allow the coupled co-expression of two genes from a singlepromoter element (FIG. 4 b). However, a person skilled in the art mayalso realize the invention by expressing a selection and/or marker genefrom a separate expression cassette contained in the retroviralconstruct, driven by an additional promoter element. For the expressionof multimeric proteins, like immunoglobulins, from separate retroviralvectors, it is preferred that different binding protein chains arelinked to different selection and/or screening markers, thereby allowingseparate monitoring for the stable transduction of the differentexpression constructs.

In case the expression of the recombinant binding protein is driven by aseparate promoter, as outlined above, any selection or screening markergene can also be cloned downstream of the 5′LTR and downstream of the5′LTR and ψ packaging signal, such that its expression is driven by the5′LTR promoter (see FIGS. 3 and 4).

As mentioned above, a preferred embodiment of the invention is toexpress recombinant antibodies or fragments thereof in cells of theB-lymphocyte lineage, preferably in preB lymphocytes. It is thereforefurther preferred to drive the expression of recombinant antibodies bypromoter and enhancer combinations that are known to confer high-levelexpression selectively in B-lineage cells. Such promoter/enhancercombinations can be e.g., but not limited to, immunoglobulin κ lightchain promoter, κ-intron and 3′κ enhancer combinations, immunoglobulinheavy chain, heavy chain intron and 3′α enhancer combinations. Thecombination of immunoglobulin κ light chain promoter, κ-intron and 3′κenhancer combinations is preferred (FIGS. 4 a,b), because it is knownthat this combination allows high level expression of immunoglobulinchains in B lineage cells and because this combination of cis-regulatorygenetic elements is able to promote somatic hypermutation to codingregions of antibodies in a regulated fashion, mediated by the activatedB cell specific enzyme AID (activation induced cytidine deaminase),which is an embodiment of the invention as detailed further below.

However, any person skilled in the art may appreciate that expression ofa particular recombinant antibody in retroviral vectors for realizingthe invention may be effected by any combination of cis-regulatorypromoter/enhancer elements and coding regions that allows expression theantibody in the desired vertebrate host cell either on the cell surfacemembrane, or in secreted form.

Although it is a preferred embodiment of this invention to expressmultimeric binding proteins, such as antibodies, from separateretroviral expression constructs (FIGS. 4 a and b), the invention mayalso be realized, if the expression of different protein chains ofmultimeric binding proteins is linked on the same retroviral expressionconstruct. In the case of immunoglobulins, this may be accomplished by,but not limited to, expression of the heavy and light chain from onepromoter and separating the coding regions for heavy and light chains byIRES sequences. In this alternative, it is preferred to clone the heavychain directly downstream of the promoter and the light chain downstreamof the IRES, because it is known that a gene following the IRES is oftenexpressed at somewhat lower levels than the gene upstream of the IRES.As light chains are the smaller molecules, it is anticipated that abetter stoichiometric expression of heavy and light chains expressed viaIRES linkage is achieved, if light chain expression is controlled viaIRES.

Alternatively, co-expression of two chains of a dimeric binding proteinsuch as an antibody may be achieved by cloning two separate expressioncassettes into a single retroviral backbone, such that the expression ofeach individual binding protein chain is separately controlled.Substitute to this approach, it is also possible to link the expressionof two different binding protein chains in the same vector by the use ofbi-directional promoters that confer transcriptional activities intoopposite directions. The latter option has the potential advantage thatpromoter interference does not occur, which may negatively affectexpression levels of promoters in close proximity.

It should be emphasized that independent from the detailed geneticorganization of retroviral vectors harbouring two binding protein codingregions, e.g., heavy and light chain of immunoglobulins, this methodallows for a single retroviral gene transfer of binding protein pairsinto target cells, which allows for facilitated control of clonalexpression of dimeric binding proteins and reduces the time-frame forthe generation of a binder expressing cell population, in comparison toa two-step retroviral transduction protocol.

In addition to cis-regulatory genetic elements, like promoters andenhancer, and selectable or screenable marker genes, like antibioticresistance markers and genes encoding auto-fluorescent proteins, thecoding regions for immunoglobulin heavy and light chains can be clonedinto the retroviral expression vectors in different contexts.

In a preferred embodiment of the invention the immunoglobulin heavy andlight chain coding regions are cloned into retroviral expressionconstructs as contiguous cDNA sequences, including leader sequencesrequired for proper surface expression and/or secretion. Examples of thebasic design for such expression vectors with enhancer elements aredepicted in FIG. 4 a. Preferably the heavy chains encode human γ1 heavychain isotypes and the light chains κ light chain isotypes, however, itis to be understood that any other heavy and light chain isotypes ofhuman or other vertebrate species may be employed in order to realizethe invention. In such retroviral cDNA expression vectors it ispreferred to include a unique restriction enzyme at the junction betweenthe variable and constant coding regions, which would allow thereplacement of only V_(H) and V_(L) coding regions in order to alter thespecificity of the expressed antibodies, or which allows the insertionof a multitude of V_(H) and V_(L) coding regions for the expression ofdiverse retroviral antibody libraries in the target cells. In apreferred embodiment the restriction enzyme site introduced at theborders of V_(H)—Cγ1 and V_(L)—C_(κ) is an Eco47III site (FIGS. 4 a, b),which does not alter the amino acid composition of the expressed heavychains and would only lead to a conserved threonine to serine amino acidchange at the first position of the constant κ coding region, which doesnot affect the binding properties of retrovirally expressed human IgG₁molecules.

As an alternative to retroviral constructs containing the codinginformation for heterologous, preferably fully human antibodies in cDNAconfiguration, retroviral expression vectors may be employed containingthe coding regions in genomic configuration, with the typicalexon-intron structure found for immunoglobulin heavy and light chains inthe germline. As retroviral vectors will be transcribed into mRNA uponretroviral particle packaging, such an organization of expressionconstructs requires that the transcriptional organization of the codingregions runs in opposite direction to the transcriptional orientation ofthe 5′LTR of the retroviral genome, because otherwise the retroviraltransfer vector would already be spliced, upon which the exon-intronstructure would be lost before transduction and stable integration ofthe recombinant construct into the target cells. However, theseconstructs offer the functionality that antibodies may be expressed aseither membrane bound or as secreted antibodies, depending on the natureof the target cell for transduction, and the ability of the target cellto either terminate transcription at the internal stop codon forsecreted antibodies, or by alternative splicing of a splice donorupstream of the stop codon for secreted antibodies, to splice acceptorsof the membrane spanning exons of membrane bound immunoglobulin.

A preferred aspect of the invention is the generation and utilization ofretroviral expression constructs for human antibodies or anyheterologous antibody or fragment thereof, in which the variable codingregion of the heavy and/or the light chain still needs to be assembledin the target cells from V, optionally D, and J gene segments in“quasi-germline” configuration by the process of V(D)J recombination.Illustrations of the basic design of such expression vectors aredepicted in FIG. 4 b, which still share the feature of thenon-rearrangeable constructs that “germline” V-D-J or V-J cassettes forheavy and light chains can be replaced by unique restriction enzymesites, including preferably Eco47III at the 3′ border of the J-elementcoding region. The V, D and J-elements contained in such vectors areflanked by conserved recombination signal sequences (RSSs) known to berecognition motifs for the recombination activating genes (RAG) 1 and 2.Upon co-expression of RAG1 and RAG2 in any vertebrate cell, such vectorswill site-specifically recombine V, optionally D and J gene segments inorder to generate the V_(H) and V_(L) regions encoding the variabledomains of antibody heavy and light chains, respectively. The expressionof RAG-1 and RAG-2 genes, thus V(D)J recombination activity, is normallyrestricted to early precursor lymphocytes. Therefore, the preferred useof precursor lymphocyte to realize the invention automatically providesthe activity for V(D)J recombination. However, it is known that uponRAG-1 and RAG-2 over-expression, any somatic vertebrate cell line can berendered proficient for V(D)J recombination, and any person skilled inthe art may therefore also realize this aspect of the invention with anynon-precursor lymphocyte cell line, by conferring ectopic expression ofRAG-1 and RAG-2. As an alternative even RAG-1 or RAG-2 deficient celllines may be employed in which the RAG-1 or RAG-2 deficiency iscomplemented by overexpression of the corresponding RAG gene or afragment thereof.

Such V(D)J rearrangeable constructs have the advantage that from asingle retroviral expression construct that is stably transduced into avertebrate host cell a diverse repertoire of antibody specificities canbe generated via RAG-1 and RAG-2 mediated V(D)J recombination.

Although it is known that the joining of V, D and J gene elementsinvolves a great degree of imprecision that contributes significantly tothe diverse amino acid sequences found in V_(H) and V_(L)complementarity determining region (CDR)₃, it is preferred to employ acollection of V-D-J-Cγ1 and V-J-Cγ retroviral construct, representingseveral V-region families, D and J elements, in order to increase thevariability already at the level of germline gene segment sequencesprovided. Nevertheless, would the preferred use of retroviral constructallowing the somatic assembly of V, optionally D, and J gene segmentsmediated by the process of V(D)J recombination allow the generation of alarge diversity of variable domain binding regions upon transductioninto precursor lymphocytes in situ, so that diverse collection of IgHand IgL chains can be generated from single or limited numbers ofconstructs.

The diversity generated by imprecise joining of V, D and J gene segmentsis greatly increased by the presence of the precursor lymphocytespecifically expressed gene terminal deoxynucleotidyl transferase (TdT),which is the only DNA polymerase that is able to add nucleotides to 3′DNA ends without a complementary template DNA strand. In order toincrease junctional diversity it is preferred to either employ cellswith high endogenous TdT expression levels, or, alternatively, toectopically express TdT in the target host cells used for retrocytedisplay, by methods known in the art.

Another embodiment of the invention is the use of V(D)J rearrangeableretroviral constructs containing more than one V, or D or J genesegment, such that by the process of V(D)J recombination different V, Dand J gene segments may be used in different rearranged clones from thesame construct. The incorporation of a multitude of different V, D and Jgene segments into such constructs is only restricted by the totalcapacity of retroviral vectors accepting DNA, which is reported to bemaximally in the range of 8-10 kilobases.

Although the employment of V(D)J recombination competent retroviralconstructs (FIGS. 4 a,b lower panels) for the expression of heterologousantibodies or fragments thereof is an aspect of the current invention,it is clear that the generation of a diverse repertoire via thisapproach is mainly restricted to the generation of diversity in the CDR3regions of immunoglobulin heavy and light chains, very similar to thecharacteristic of a primary antibody repertoire generated during early Blymphopoiesis.

A hallmark of the adaptive immune system is its capability of affinitymaturation of antibody variable domains, which is based on the somatichypermutation of variable domain coding regions. Somatic hypermutationis known to be strongly enhanced by the enzyme activation inducedcytidine deaminase (AID). High-level somatic hypermutation additionallydepends on the presence of cis-regulatory enhancer elements from theimmunoglobulin gene locus, and a beneficial effect has most clearly beendescribed for combinations of the Igκ intron and 3′κ enhancer elements.An aspect of the current invention is therefore the employment ofretroviral expression constructs containing these cis-regulatoryelements to retrovirally express such immunoglobulin expressionconstructs in target cells endogenously or ectopically expressing theAID enzyme, either constitutively or inducibly, by methods known in theart.

The application of ‘Retrocyte Display’ in the context of somatichypermutation competent retroviral constructs and in the context of AIDexpressing host cells allows for a further diversification of anantibody in situ, after transduction into AID expressing host cells.

The combination of these aspects of the invention recapitulates allmolecular and genetic events occurring in the adaptive immune system,namely the generation of a primary antibody repertoire from one or alimited number constructs comprising a limited number of V, D and J genesegments and the additional AID-mediated somatic hypermutation of thecoding regions for antigen-binding variable domains of antibodies.

The specific selection of higher affinity antibody binders to desiredantigens can be accomplished by Retrocyte Display via increased bindingto desired antigens of choice detected by standard FACS basedtechnology, followed by high-speed preparative cell sorting of strongantigen binders. Strong binders can thus be selectively isolated, andthe antibody genes encoded by the retroviral vectors can be re-isolated,cloned and sequenced from selected cells or cell clones by standardmolecular biology methods, known in the art, including, but not limitedto genomic and RT-PCR.

In a preferred embodiment the final cell sorting step is performed as asingle cell sort, allowing the clonal isolation and final expansion ofantigen reactive cell clones, which facilitates cloning and sequencedetermination of the coding region of cognate IgH and IgL chain pairsfrom selected binders.

If desired, FACS-enriched cells can be expanded in culture and canoptionally again be subjected to antigen binding and repeated high-speedcell sorting of highly reactive cells, a process that can optionally beapplied repeatedly, until desired staining intensity and hence expectedbinding specificity for a desired antigen is achieved (FIG. 1). Thisselective enrichment and in vitro expansion of antigen reactive cellsmimic the selective outgrowth of higher affinity binders occurring in Tcell dependent immune reactions.

It should be noted that high-speed cell sorter assisted enrichment ofantigen-reactive cells is only a preferred method of realizing theinvention, but that other ways of selecting and isolating cells forantigen reactivity, like for instance, but not limited to, panningmethods, where cells are bound to immobilized antigens on a solidsupport, may also be applied. Furthermore, it is possible to enrichantigen-reactive cells by micromanipulative approaches, e.g., but notlimited to, growing cells under limiting dilution conditions inmicrotiter plates or as cell clones in semi-solid medium, which allowsspecific antigen-staining and/or labelling of cell clones and theiridentification by microscope assisted ways followed by manual and/orroboter assisted picking of antigen reactive clones.

A further embodiment of the invention is to perform repetitive cycles ofantigen-selection/FACS-sorting/expansion of antigen-reactive cells inthe presence of mutagenizing conditions, specifically targetingmutations to the coding regions of variable antibody binding domains. Bythis approach higher affinity mutants, which are generated in situ, aregenerated in each round of cell amplification. Upon cell sorting andenrichment of cells showing increased antigen binding upon retrocytedisplay, higher affinity mutants can selectively be enriched andexpanded. A high mutation rate targeted to antibody variable regiondomains can be achieved by overexpressing the AID enzyme in the antibodyexpressing cells, in particular, when the expression constructs containcis-regulatory promoter and enhancer elements, including, but notlimited to immunoglobulin κ intron and 3′κ enhancer elements, that areknown to confer AID mediated somatic hypermutation to antibody variableregions (FIGS. 4 a and b). While such an approach could be realizedusing cells that constitutively express AID, either endogenously orectopically, one aspect of the invention utilises AID expressionvectors, in which AID expression can be induced and again switched offusing inducible promoters, e.g., but not limited to, tetracycline and/ordoxycycline inducible promoter systems (Gossen & Bujard, 1992), in whichthe expression of a gene of interest is controlled by a minimal CMVpromoter flanked by tandem repeats of the prokaryotic tet-operon, andwhich can be induced or suppressed for expression using aHSV-VP16-Tet-repressor fusion protein, whose binding to the tet-operonis allosterically controlled by tetracycline or tetracyclinederivatives.

In the following non-limiting examples, the present invention isexplained in more detail.

Example 1 Cloning of Retroviral Expression Vectors for Fully HumanImmunoglobulin Heavy (IgH) and Immunoglobulin Light (IgL) ChainsContaining HygromycinB and Puromycin Antibiotic Drug Selection Markers,Respectively

As mentioned before, the invention can be realized with retroviralexpression vectors for binding proteins of different design (comparee.g. FIGS. 4 a-c). As an example of one of the vector designs that canbe used to realize the invention, the detailed cloning strategy forretroviral expression vectors is described herein allowing theexpression of fully human IgG₁/κL antibodies, and the selection for thestable maintenance of these vectors in target cells using antibioticresistance markers.

a) Construction of retroviral expression vectors for humanimmunoglobulin heavy (IgH) chains

As a starting point for construction of retroviral human immunoglobulinheavy expression vectors, the commercially available retroviral vectorpLHCX was used (BD-Clontech, Mountain View, Calif.) (FIG. 5 a). pLHCXcontains an hygromycinB resistance marker gene driven by the 5′LTRpromoter of the retroviral backbone. In addition, pLHCX contains theCMV-immediate early promoter followed by simple multiple cloning site(MCS) for insertion of genes of interest to be expressed. In addition,the pLHCX backbone contains a convenient unique BglII restriction enzymesite upstream of the CMV promoter (FIG. 5 a), into which additionalgenetic elements can be cloned.

It is a preferred embodiment of the invention to use the Eco47IIIrestriction enzyme for in-frame cloning of human V_(H) coding regions tothe human constant γ1 heavy chain coding regions, as this particularrestriction enzyme site can be introduced at the junction between V_(H)and Cγ1 coding regions without changing the amino acid composition ofexpressed IgH chains. However, pLHCX contains one Eco47III restrictionenzyme site in the ψ packaging signal (FIG. 5 a) that would preclude thestraightforward use of Eco47III for the above-mentioned V_(H) regioncloning strategy. In order to remove this inconvenient Eco47IIIrestriction enzyme site from the pLHCX vector backbone, the followingfirst preparatory cloning step was performed as detailed in FIG. 5 a.The Eco47III site in the ψ packaging signal was removed by site-directedmutagenesis using a commercial Quikchange™ kit (Stratagene, La Jolla,Calif.) replacing the third C nucleotide of the Eco47III recognitionsequence AGCGCT with an A, using specific primer pairs conferring thedesired mutation according to the instructions of the manufacturer. Themodified vector was designated pLHCX-m1 and it was verified that thissingle-basepair substitution in the ψ (Psi) packaging signal did notaffect the retroviral transduction efficiency of the modified vectorpLHCX-m1 (data not shown).

Into the pLHCX-m1 backbone, cDNAs encoding the constant region for humanCγ1 either with or without membrane spanning coding regions M1 and M2have been cloned in parallel. The Cγ1-m and Cγ1-s DNA fragments wereamplified by RT-PCR using cDNA of human peripheral blood lymphocytes astemplate and forward and reverse primers Seq-ID1, Seq-ID2, Seq-ID3 (seebelow). For the RT-PCR amplification of the membrane bound form of humanIgG, the primer combination Seq-ID1 and Seq-ID2 was used and for thecloning of the secreted version of human IgG, the primer combinationSeq-ID1 and Seq-ID3 was used. The forward and reverse PCR amplificationprimers contained HindIII and ClaI restriction enzyme sites,respectively, allowing directional cloning of the PCR-amplifiedfragments into the unique HindIII and ClaI sites downstream of the CMVpromoter in pLHCX-m1 (FIG. 5 a). The forward PCR amplification primeradditionally contained an internal Eco47III site useful for in-framefusion of V_(H) regions to the constant regions, without changing theamino acid composition of the expressed full-length IgG₁ heavy chains.The reverse PCR amplification primers Seq-ID2 and Seq-ID3 containedadditional internal NotI sites, which allowing the restriction enzymedigestion of the construct directly downstream of the coding region, forgeneral cloning purposes, e.g. the exchange of the constant regioncoding region for the expression of different Ig isotypes.

Seq-ID1: 5′-GATC AAGCTTAGCGCT TCCACCAAGGGCCCATCGGTCTTCCC-3′       HindIII/Eco47III

The primer Seq-ID2 was used as a reverse primer for PCR amplification ofthe secreted version of human IgG₁ together with Seq-ID1, and containeda unique NotI site (underlined) for cloning purposes.

Seq-ID2: 5′-GATC ATCGATGCGGCCGC TCATTTACCCGGAGACAGGGAGAGG-3′          claI/NotI

The primer Seq-ID3 was used as a reverse primer for PCR amplification ofthe membrane bound version of human IgG₁ together with Seq-ID1, andcontained a unique NotI site (underlined) for cloning purposes.

Seq-ID3: 5′-GATC ATCGATGCGGCCGC TAGGCCCCCTGCCTGATCATGTTC-3′         ClaI/NotI

The resulting PCR-products of ca. 1.0 kb for the secreted version ofhuman Cγ1 and of ca. 1.2 kb for the membrane bound version of human Cγ1were digested with HindIII and ClaI restriction enzymes and were inparallel directionally cloned into the compatible restriction enzymesites pLHCX-m1, resulting in plasmids pLHCX-m1-Cγ1-s and pLHCX-m1-Cγ1-m,respectively (see also FIG. 5 b). V_(H) chain regions could then becloned in-frame to the coding regions for secreted or membrane-boundhuman Cγ1 using unique restriction enzymes HindIII and Eco47III,flanking V_(H) region fragments (FIG. 5 b). This combination ofrestriction enzymes is only very rarely found in human V_(H) codingregions of all 7 human V-gene segment families.

In order to construct a complete human IgG1 heavy chain expressionvector, a human V_(H) coding region from a previously identified fullyhuman antibody, specific for NIP-Ovalbumin, was inserted into theconstructs pLHCX-m1-Cγ1s and pLHCX-m1-Cγ1m as a HindIII-Eco47IIIfragment resulting in plasmids pLHCX-m1-VHCγ1s and pLHCX-m1-VHCγ1m,respectively (FIG. 5 c). The V_(H) coding region for the NIP-Ovalbuminspecific human antibody including leader sequence and 5′-HindIII and3′Eco47III cloning sites is provided in Seq-ID4. It should be noted thattwo additional C nucleotides had been added upstream of the start-ATGfor improved translation (approximation of a Kozak-consensus sequence):

Seq-ID4: AAGCTT CCATGGAGTTTGGGCTcAGCTGGGTTTTCCTTGTTGCTCTTTTAAGAGGTGTCCAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGIGGGIGGCAGTTATATCATATGATGGAAGCAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAATGGTCGACCACGCGGAAAGCTACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACAA TGGTCACCGTCTCT AGCGCT

HindIII and Eco47III restriction enzyme sites for cloning are underlinedin Seq-ID4. The start ATG of the leader sequence starts at position 9.

While this human IgG₁ heavy chain expression vectors depicted in FIG. 5c are already sufficient to realize the invention and to performretrocyte display in combination with retroviral IgL chain expressionvectors, the functionality of targeting somatic hypermutation to V_(H)coding regions requires the presence of certain cis-regulatory enhancerelements of the immunoglobulin light or heavy chain gene loci. As the κintron and 3′κ enhancer elements of the κ light chain gene locus areknown to be capable of targeting somatic hypermutation to V regionslocated downstream of an active promoter, the basic retroviral human Igheavy chain expression vectors pLHCXm1-VHCg1m and pLHCXm1-VHCg1s (FIG. 5c) have additionally been modified to contain κ intron and 3′κ enhancerelements in the following way. The sequence of the murine κ intronenhancer (κiE) is located within a ca. 2.3 kb long intergenic regionlocated between the Jκ5 element and the constant κ coding region, whosesequence can de derived from NCBI-Genbank entry V00777. The core κiEcomprises only about 0.5 kb within this intergenic region and itssequence can be derived from NCBI Genbank entry X00268. The entire 2.3kb fragment from Jκ5 to Cκ, including the κiE region, contains aninternal BglII site, precluding the use of this restriction enzyme forcloning of a PCR amplified genomic fragment into the pLHCX-m1-VHCγ1-sand pLHCX-m1-VHCγ1-m vectors. However, there is no internal BamHIrestriction enzyme fragment in this region, therefore allowing thecloning of a genomic PCR fragment flanked by BamHI site into BglIIlinearized vectors pLHCX-mod 1-VHCγ1s and pLHCX-m1-VHCγ1m (FIG. 5 c).Vectors have been constructed containing both the entire ca. 2.3 kbintergenic region between Jκ5 to Cκ by PCR amplification of this genomicfragment from mouse genomic DNA using forward and reverse primersSeq-ID5 and Seq-ID6, both containing additional BamHI restriction enzymesites (underlined) for cloning into the unique BglII restriction enzymesite of pLHCX-m1-VHCγ1s and pLHCX-m1-VHCγ1m, resulting in plasmidspLHCX-m1-VHCγ1s-κiE and pLHCX-m1-VHCγ1m-κiE, respectively (FIG. 5 d).

Seq-ID5: 5′-GATC GGATCC GTACACTTTTCTCATCTTTTTTTATGTG-3′         BamHISeg-ID6: 5′-GATC GGATCC CTGAGGAAGGAAGCACAGAGGATGG-3′         BamHI

In addition to inserting the entire ca. 2.3 kb κiE containing genomicfragment from the mouse κ light chain gene locus, also a shorter, ca.0.8 kb genomic PCR fragment (position 3634-4394 of V00777, Seq-ID7),containing the core κiE has been cloned into the unique BglII site ofpLHCX-m1-VHCγ1-s and pLHCX-m1-VHCγ1-m (not shown). The forward andreverse PCR primers used for PCR amplification of this genomic DNAfragment are depicted in Seq-ID8 and Seq-ID9.

Seq-TD7: 5′-GAAAAATGTTTAACTCAGCTACTATAATCCCATAATTTTGAAAACTATTTATTAGCTTTTGTGTTTGACCCTTCCCTAGCCAAAGGCAACTATTTAAGGACCCTTTAAAACTCTTGAAACTACTTTAGAGTCATTAAGTTATTTAACCACTTTTAATTACTTTAAAATGATGTCAATTCCCTTTTAACTATTAATTTATTTTAAGGGGGGAAAGGCTGCTCATAATTCTATTGTTTTTCTTGGTAAAGAACTCTCAGTTTTCGTTTTTACTACCTCTGTCACCCAAGAGTTGGCATCTCAACAGAGGGGACTTTCCGAGAGGCCATCTGGCAGTTGCTTAAGATCAGAAGTGAAGTCTGCCAGTTCCTCCAAGGCAGGTGGCCCAGATTACAGTTGACCTGTTCTGGTGTGGCTAAAAATTGTCCCATGTGGTTACAAACCATTAGACCAGGGTCTGATGAATTGCTCAGAATATTTCTGGACACCCAAATACAGACCCTGGCTTAAGGCCCTGTCCATACAGTAGGTTTAGCTTGGCTACACCAAAGGAAGCCATACAGAGGCTAATATCAGAGTATTCTTGGAAGAGACAGGAGAAAATGAAAGCCAGTTTCTGCTCTTACCTTATGTGCTTGTGTTCAGACTCCCAAACATCAGGAGTGTCAGATAAACTGGTCTGAATCTCTGTCTGAAGCATGGAACTGAAAAGAATGTAGTTTCAGGGAAGAAAGGCAATAGAAGGAAGCCTGAGAATATCTTCAAAGGG-3′ Seq-ID8: 5′-GATC GGATCCGAAAAATGTTTAACTCAGCTAC-3′         BamHI Seq-ID9: 5′-GATC GGATCCCCCTTTGAAGATATTCTCAGGCTTCC-3′         BamHI

The ca. 0.8 kb fragment core κiE containing genomic PCR fragment wasalso cloned as a BamHI digested PCR fragment both into the unique BglIIrestriction enzyme site of vectors pLHCX-m1-VHCγ1-s and pLHCX-m1-VHCγ1-m(not shown here).

The sequence of the murine 3′κ enhancer element deposited can beretrieved under NCBI-Genbank reference number X15878, and is containedin an 808 bp gene sequence located ca. 8.7 kb downstream of the constantκ coding region in the mouse genome.

The murine 3′κ enhancer does not contain an internal ClaI site and wastherefore PCR-amplified from mouse genomic DNA using forward and reversePCR primers Seq-ID10 and Seq-ID11, respectively, containing additionalClaI restriction enzyme sites for cloning into the unique ClaI site ofretroviral vectors pLHCX-m1-VHCγ1s-3′κ E and pLHCX-m1-VHCγ1m-3′κ E (FIG.5 d).

Seq-ID10: 5′-GAGA ATCGAT AGCTCAAACCAGCTTAGGCTACAC-3′                  ClaI Seq-ID11: 5′-GAGA ATCGATTAGAACGTGTCTGGGCCCCATG-3′                   ClaI

This resulted in the final Igγ₁H chain expression vectorspLHCX-m1-VHCγ1s-3′κE-κiE and pLHCX-m1-VHCγ1m-3′κE-κiE (FIG. 5 e)encoding either Ig heavy chains that, upon IgL chain co-expression, leadto the production of secreted or to membrane bound human IgG₁antibodies, respectively.

Both vectors additionally contain κiE and 3′κE cis regulatory elementsupstream and downstream of the Igγ₁H chain expression cassette,conferring somatic hypermutation to the V_(H) regions of the expressedIgγ₁H chains.

b) Cloning of retroviral expression vectors for human Igκ light chains

As a starting point for construction of retroviral human immunoglobulinlight chain expression vectors allowing antibiotic selection forretroviral integration, the commercially available retroviral vectorpLPCX (BD-Clontech, Mountain View, Calif.) has been used (FIG. 6 a).This vector contains an antibiotic selection marker conferring puromycinresistance, driven by the 5′LTR promoter of the retroviral backbone.Although similar in design as the pLHCX backbone (see Example 1a), pLPCXcontains two Eco47III sites and a MCS with more restriction enzymesites, but lacks the convenient unique BglII site upstream of the CMVpromoter (FIG. 6 a).

In order to remove the Eco47III restriction enzymes from the pLPCXvector backbone and at the same time to introduce a unique BglIIrestriction enzyme upstream of the CMV promoter, the followingpreparatory cloning steps were performed: In a first step, the Eco47IIIsites in the packaging signals of pLHCX was removed by site-directedmutagenesis using a commercial Quikchange™ kit (Stratagene, La Jolla,Calif.) replacing the third C nucleotide of the Eco47III recognitionsequence AGCGCT with an A, using specific primer pairs conferring thedesired mutation according to the instructions of the manufacturer (FIG.6 a). It was verified that this single-base pair substitution in the ψ(Psi) packaging signal did not affect the retroviral transductionefficiencies of the mutated vectors (data not shown). The mutated vectorwas designated pLPCX-m1 (FIG. 6 a). In order to obtain a pLPCX vectorbackbone completely devoid of Eco47III sites and additionally includinga unique BglII site upstream of the CMV promoter, an AscI-NcoI fragmentfrom pLPCX-m1, in which the NcoI digested DNA end had been filled-in byKlenow enzyme, was cloned into an AscI-BlpI digested pLHCX backbone, inwhich the BlpI digested DNA end had been filled in by Klenow enzyme(FIG. 6 b), thereby generating a vector designated pLPCX-m2, in whichessentially only the hygromycinB gene of pLHCX had been replaced by thepuromycin resistance marker of pLPCX (FIG. 6 b).

For the construction of the IgκL chain expression vector, the constant κlight chain coding region was PCR cloned from human peripheral bloodlymphocyte cDNA using forward and reverse primers Seq-ID12 and Seq-ID13,containing HindIII and ClaI restriction enzyme sites, respectively fordirectional cloning into pLPCX-m2 (FIG. 6 b). As described under sectiona.), the forward primer Seq-ID12 additionally contained an Eco47III siteallowing in-frame fusion of V_(L) coding regions to the constant κ lightchain coding region, only resulting in one conserved threonine to serineamino acid substitution at the first position of the human constant κlight chain. The reverse primer contained an additional internal NotIsite to facilitate later cloning procedures, like e.g. the exchange ofthe constant κ coding region.

Seq-ID12: 5′-GATC AAGCTTAGCGCT CTGTGGCTGCACCATCTGTCTTCATC-3′       HindIII/Eco47III Seq-ID13: 5′-GATC ATCGATGCGGCCGCCTAACACTCTCCCCTGTTGAAGCT-3′         ClaI/NotI

The insertion of the constant κ light chain coding region flanked byHindIII/Eco47III sites at the 5′ end and NotI/ClaI sites at the 3′ endinto pLPCX-m2 resulted in plasmid pLPCX-m2-Cκ.

In order to construct a complete human IgκL heavy chain expressionvector, a human Vκ coding region from a previously identified fullyhuman antibody, specific for NIP-Ovalbumin, was inserted into theconstruct pLPCX-m2-Cκ as a HindIII-Eco47III fragment (FIG. 6 c). The Vκcoding region for the NIP-Ovalbumin specific human antibody includingleader sequence and 5′-HindIII and 3′Eco47III cloning sites is providedin Seq-ID14. It should be noted that two additional C nucleotides hadbeen added upstream of the start-ATG for improved translation(approximation of a Kozak-consensus sequence):

Seq-ID14: 5′- AAGCTT CCATGGATATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTGCCAGATGTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGCGCAACTCACACCATTACCAGCTATTTAAATTCCTATCACCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTcAACAGAGTTACAGTACCCCCACTTTCGGCCAAGGGACCAAGGTGGAAATCA AGCGC T -3′

HindIII and Eco47III restriction enzyme sites for cloning are underlinedin Seq-ID14. The start ATG of the leader sequence starts at position 9.Insertion of this HindIII-Eco47111 fragment into HindIII-Eco47IIIlinearized pLPCX-m2-Cκ resulted in expression construct pLPCX-m2-VκCκ(FIG. 6 c).

While this retroviral κ light chain expression vector is alreadysufficient to realize the invention and to perform retrocyte displayupon co-expression with retroviral Ig heavy chain expression vectors,additional vectors have been cloned also containing κiE and 3′κEelements, following the same cloning strategy as for the Ig heavy chainexpression constructs. Thus, the mouse κiE was inserted into the uniqueBglII site in pLPCX-m2-Vκ-Cκ, upstream of the CMV promoter, either as aca. 2.3 kb genomic, BamHI digested PCR fragment amplified with primerpairs Seq-ID5 and Seq-ID6 (see above), or as a ca. 0.8 kb genomic, BamHIdigested PCR fragment amplified with primer pairs Seq-ID8 and Seq-ID9(see above). Only the cloning of the ca. 2.3 kb genomic mouse κiEcontaining fragment into pLPCX-m2-VκCκ is depicted here, resulting inplasmid pLPCX-m2-VκCκ-κiE (FIG. 6 d).

Finally, and in analogy to the construction κiE and 3′κE containing IgHchain retroviral expression vectors described in Example 1a above, themurine 3′κE was inserted as a ClaI digested genomic PCR fragmentamplified with primer pairs Seq-ID10 and Seq-ID11 into the unique ClaIrestriction site downstream of the κ light chain coding region in orderto generate the retroviral expression vector pLPCX-m2-Vκ-Cκ-κiE-3′κE(FIG. 6 d).

Like the IgH chain expression vector containing κiE and 3′κE elements,this vector now contains all cis-regulatory elements required to confersomatic hypermutation to any Vκ coding region cloned into the construct(see below).

Example 2 Generation of a Cell Line Over-Expressing Activation InducedCytidine Deaminase (AID)

It has been demonstrated that the activated B cell specific proteinactivation induced cytidine deaminase (AID) is a unique trans-activatingfactor that is required and sufficient to confer a somatic hypermutationphenotype to any vertebrate cell line. In cells expressing AID, somatichypermutation can specifically be targeted to transcriptionally activegene loci, if they are arranged in correct context to cis-regulatoryenhancer elements, in particular κiE and 3′κE elements of theimmunoglobulin κ light chain locus. In order to obtain cell lines stablyexpressing AID, first, a retroviral expression construct encoding murineAID was constructed in the following way:

The murine AID cDNA was PCR amplified using high-fidelity Pfx-polymerase(Invitrogen, Carlsbad, Calif.) from total mouse spleen cDNA according tothe instruction of the manufacturer, using forward and reverse PCRprimers Seq-ID15 and Seq-ID16, containing additional XhoI cloning sitesfor ligation of the PCR amplified fragment into compatible vectors. Inaddition, the forward primer contained additional two C nucleotides(highlighted in italics) downstream of the XhoI site and upstream of thestart ATG codon of the murine AID ORF, in order to approximate a Kozaktranslational initiation sequence and thereby ensuring propertranslation of the cloned cDNA.

Seq-ID15: 5′-AATA CTCGAG CCATGGACAGCCTTCTGATGAAGCAAAAG-3′         XhoISeq-ID16: 5′-AATA CTCGAG TCAAAATCCCAACATACGAAATGCATC-3′         XhoI

The resulting 620 bp RT-PCR product was digested with XhoI and wasligated into XhoI digested and alkaline phosphatase treated pLPCX, fromBD-Clontech (Mountain View, Calif.). Ligation products containing theinsert in correct orientation were determined by diagnostic restrictionenzyme digestion. A clone with correct restriction enzyme patterncontaining the murine AID cDNA insert in correct orientation wasverified by DNA sequencing and was designated pLPCX-mAID and (FIG. 7).

The sequence of the murine AID cDNA cloned corresponded exactly to thepublished murine AID cDNA ORF provided in NCBI-Genbank entry AF132979.

Next, 10 μg of PvuI linearised pLPCX-mAID construct was transfected into5×10⁶ FA-12 Abelson transformed preB cells resuspended in 800 μl plainRPMI medium by electroporation at 300V, 960 μF at ambient temperature.Transfected cells were resuspended in 20 ml growth medium containing FCSand were plated into ten 96 well plates at 200 μl/well. 48 hours posttransfection, stably transfected cells were selected by adding 2 μg/mlpuromycin antibiotic to the growth medium.

After 10-14 days post transfection, dozens of puromycin resistantcolonies were detectable and selected clones were transferred into freshculture medium containing 2 μg/ml puromycin. The puromycin resistantclones were further expanded and a selected number of clones were testedfor expression of murine AID protein by ECL Western-blotting using acommercial anti-mouse AID antibody as recommended by the manufacturer(see FIG. 9 a).

A specific AID protein band was detectable in ca. 80% of the analyzedFA-12-AID transfected cell clones and displayed an apparent molecularweight of 25 kD, as expected. From this it was concluded that severalcell lines were obtained constitutively over-expressing the murine AIDprotein.

Example 3 Demonstration of Somatic Hypermutation Targeted to a ReporterGene in Retroviral Human Immunoglobulin Expression Constructs ContainingCis-Regulatory κiE and 3′κE Elements

Next, it was demonstrated, that human antibody variable regions inretroviral expression constructs, as disclosed in this invention, aretargets for AID mediated somatic hypermutation. For this, a reporterconstruct was generated, in which the V-region ORF of a human IgH chainwas replaced with a mutated EGFP ORF, in which a stop codon had beenintroduced in the context of a RGYW sequence motif that is known to be ahotspot for somatic hypermutation (Bachl & Olsson, 1999).

The stop mutation was introduced at codon 107 of the EGFP ORF changing atyrosine codon to a TAG stop codon. In addition, codon 108 was modified,thereby generating a novel diagnostic SpeI restriction site within inthe mutated EGFP sequence, such that upon reversion of the stop-mutationin codon 107 the SpeI site would be destroyed, thereby facilitating theidentification and characterization of revertants. The sequencemodification introduced into the EGFP ORF is depicted in FIG. 10 a, theentire mutated EGFP ORF is provided in FIG. 10 b.

The reporter construct for demonstrating somatic hypermutation wasconstructed as follows:

The EGFP ORF was PCR amplified from plasmid pIRES-EGFP (BD-Clontech,Mountain View, Calif.) as a template with high-fidelity Pfx-Polymerase(Invitrogen, Carlsbad, Calif.) and forward primers Seq-ID17 andSeq-ID18, each containing additional HindIII and Eco47III restrictionenzyme sites allowing replacement of the V_(s)-region inpLHCXm1-VHCγ-s-κiE-3′κE with a EGFP ORF. The forward primer containedadditional two C nucleotides upstream of the ATG start-codon,highlighted in italics, which approximates a Kozak translationinitiation consensus sequence and ensures proper translationalinitiation at the correct ATG start codon.

Seq-ID17: 5′-CGC AAGCTT CCATGGTGAGCAAGGGCGAGGAGCTGTTC-3′       HindIIISeq-ID18: 5′-TAG AGCGCT CTTGTACAGCTCGTCCATGCCGAGAGTG-3′       Eco47III

The Pfx amplified EGFP PCR fragment of 737 bp was directly cloned intothe pCR4-Topo vector, which is part of a Zero-Blunt PCR cloning kit(Invitrogen, Carlsbad, Calif.) resulting in the pCR4-Topo-EGFP vector(FIG. 11). Next, a sequence-verified clone of pCR4-Topo-EGFP was mutatedat codons 107 and 108 of the EGFP ORF as depicted in FIG. 10 using aQuikchange™ kit (Stratagene, La Jolla, Calif.) according to themanufacturer's instructions using specific primer pairs conferring thedesired mutations, thereby generating plasmid pCR4-Topo-EGFPmut.

The sequence verified, mutated EGFP ORF of pCR4-Topo-EGFPmut wasrecovered from the plasmid by double restriction enzyme digestion usingrestriction enzymes HindIII and Eco47111. The digested fragment wasligated into HindIII and Eco47III double digested plasmidpLHCXm1-VHCγ-s-κiE-3′κE (FIG. 5 e) and into HindIII and Eco47III doubledigested plasmid pLHCXm1-VHCγ-s (FIG. 5 c), not containing enhancerelements. Thereby, in both vectors the V_(H) coding regions werereplaced with the mutated EGFP ORF, which were fused in-frame to the Cγ₁regions, resulting in reporter plasmid pLHCXm1-E(mut)-Cγ-s-κiE-3′κE, andin control reporter plasmid pLHCXm1-E(mut)-Cγ-s (FIG. 11).

Both plasmids were transduced into puromycin-resistant FA-12 AIDtransfectant clones 3 (AID expressing) and 5 (no AID expression) ascontrol. Transduced cells were cultured under 2 mg/ml hygromycin Bselection beginning 24 hours after transduction, and cells were analyzedfor emergence of green auto-fluorescent cells after 6, 8 and 10 days ofculture. Only in the experiment, in which a mutated EGFP reporterconstruct was expressed in the context with κiE and 3′κE (i.e. usingplasmid pLHCXm1-E(mut)-Cγ-s-κiE-3′κE) and in FA-12 AID transfectant, inwhich AID expression was detectable by Western-blotting (i.e. FA-12 AIDtransfectant clone3), could green auto fluorescent cells be detectedafter 6, 8 and 10 days of culture, when a steady-state frequency of ca.0.2% (FIG. 9 b) green cells were detectable by FACS. In none of thecontrol experiments (no AID expression, and/or no enhancer elementspresent in the constructs, data not shown), could green cells bedetected within the 10 days duration of the experiment.

From the 0.2% EGFP positive population, 192 single cells were sortedinto individual wells of two 96 wells, and 100 clones from thesesingle-sorted clones were analyzed by FACS for green fluorescence aftersufficient cells had been grown up. From 100 clones analyzed, 95 clonesdisplayed a homogenous fluorescence pattern, similar in intensity as thefluorescence detected in the single-sorted cells, i.e. at ca. 10² logfluorescence (auto fluorescence of FA-12 cells control cells remainedbelow the 10¹ log fluorescence levels, indicated by the threshold-line).4 clones displayed a heterogeneous fluorescence pattern with ca. half ofthe cells being negative and half of the cells being positive for EGFPexpression. Only one out of 100 clones analyzed displayed practically noEGFP fluorescence, although also this clone was slightly abovebackground auto-fluorescence levels. The 5 clones with heterogeneous andnegative EGFP pattern could be due to (partial) positional silencing ofEGFP expression of retroviral integrants, or the results could be due tosingle cell sorting artefacts. Nevertheless, in the majority of clones(95%) EGFP expression was clearly detectable. 24 of these clones wereanalyzed by PCR using the cloning primers Seq-ID17 and Seq-ID18 in orderto re-amplify the EGFP gene from the stably transduced cells.

In contrast to a PCR product from the reporter vector containing themutated EGFP ORF, none of the 24 PCR products from EGFP expressingclones could be digested with SpeI restriction enzyme, suggesting areversion of the TAG stop mutation in codon 107 of the mutated EGFP ORF(data not shown).

Ten of the PCR products have been analyzed by DNA sequencing, confirmingthat all of the ten clones contained a G->C mutation of the G nucleotidein the RGYW motif introduced into the EGFP ORF, as described before inthe literature (Bachl & Olsson, 1999).

This demonstrates, that dependent on the presence of cis-regulatorygenetic elements, like κiE and 3′κE elements, and dependent on AIDexpression, elevated levels of somatic mutation, and thus mutagenesis,can be targeted to the DNA regions downstream of an active promoter,and, thus, to the V_(H) coding regions of human antibody chains in thecontext of the disclosed retroviral expression constructs.

In terms of estimating the level of AID dependent somatic mutation inthe κiE and 3′κE constructs, the estimated mutation rate was in therange of ca. 3×10 mutations/bp/generation. This value is still lower assomatic hypermutation rates that have been reported in vivo which canreach rates of up to 10⁻⁴ or even 10⁻³/bp/generation. Nevertheless, thedetected mutation rate was still significantly higher than thebackground mutation rate reported in vertebrate cells that is estimatedto be in the range of 10⁻⁸ mutations/bp/generation. Thus, it isconcluded that high somatic mutation rates are specifically targeted toregions downstream of an active promoter in the disclosed retroviralconstructs in an enhancer and AID dependent fashion, thereby allowingthe application of Retrocyte Display under in vivo mutagenizingconditions based on somatic hypermutation mediated by AID expression.

Example 4 Demonstration of In Situ Generation of Human Antibody EncodingRegions by Using V(D)J Recombination Competent Retroviral ExpressionVectors

a) Cloning of a Retroviral Human Heavy (IgH) Chain Expression VectorRequiring V(D)J Recombination Prior to IgH Chain Expression.

As an alternative approach to retrovirally expressing heavy (H) andlight (L) chains from cDNA expression vectors described in Example 1, adifferent retroviral IgH chain vector class has been constructed, inwhich the variable coding region is encoded by separate V, D and J genesegments in “quasi-germline” configuration, that still need to beassembled by the process of V(D)J recombination prior to expression.V(D)J recombination mediates site specific, but slightly impreciseassembly of V, D and J gene segments, such that diverse V coding regionscan be generated from a single expression construct upon transductioninto V(D)J recombination active cells in situ, like e.g. precursor Bcells.

For this, germline V_(H)3.30, D_(H)1.26 and J_(H)3 gene segments havebeen PCR cloned individually from genomic DNA derived from B celldepleted human peripheral blood mononuclear cells (PBMCs). The PCRprimers used for the amplification of the germline V, D and J genesegments were chosen such that flanking DNA sequences comprisingconserved recombination signal sequences (RSSs) and additionalintervening DNA sequences were included allowing proper assembly of V, Dand J gene segments. All PCR amplicons were generated using proofreadingthermostable DNA polymerase Pfx (Invitrogen, Carlsbad, Calif.) and wereinitially subcloned into a pSC-B PCR cloning vector (Stratagene, LaJolla, Calif.), in both cases according to the instruction of thesuppliers. PCR fragments, subcloned into pSC-B, were verified by DNAsequencing and fragments were only used for further cloning, if the DNAsequence had been sequence verified.

For the PCR amplification of a germline human V_(H)3.30 fragment, DNAprimers Seq-ID19 and Seq-ID20 were used containing BamHI and NheIrestriction enzyme sites (as indicated) allowing further subcloning ofthe PCR cloned DNA fragments.

Seq-ID19: 5′-ATTT GGATCC CACCATGGAGTTTGGGCTGAGCTGGGTTTTCCTCG-3′                  BamHI Seq-ID20: 5′-CCC GCTAGCTCCTGACAGGAAACAGCCTCCATCTGCACCT-3′                  NheI

This way a PCR amplicon of 623 bp length containing the germline VH3.30gene segment with flanking DNA was obtained (Seq-ID21), see FIG. 11 a.

Seq-ID21: 5′attt GGATCC CACCATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTGATTCATGGAGAAATAGAGAGACTGAGTGTGAGTGAACATGAGTGAGAAAAACTGGATTTGTGTGGCATTTTCTGATAACGGTGTCCTTCTGTTTGCAGGTGTCCAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTAGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGACACAGTGAGGGGAAGTCATTGTGCGCCCAGACACAAACCTCCCTGCAGGAACGCTGGGGGGAAATCAGCGGCAGGGGGCGCTCAGGAGCCACTGATCAGAGTCAGCCCTGGAGGCAGGTGCAGATGGAGG CTGTTTCCTGTCAGGAGCTAGC ggg3′

Next, PCR amplication of a human genomic DNA fragment containing thehuman DH1.26 fragment with flanking genomic DNA was achieved usingprimer pair Seq-ID22 and Seq-ID23, containing sites for restrictionenzymes NheI and XhoI, respectively (FIG. 11 a).

Seq-ID22: 5′-GGA GCTAGC GGGCTGCCAGTCCTCACCCCACACCTAAGGT-3′        NheISeq-ID23: 5′-GGG CTCGAG TCCTCACCATCCAATGGGGACACTGTGGAGC-3′        XhoI

This way a PCR amplicon of 336 bp length containing the germline DH1.26gene segment with flanking DNA was obtained (Seq-ID24).

Seq-ID24: 5′gga GCTAGC GGGCTGCCAGTCCICACCCCACACCTAAGGTGAGCCACAGCCGCCAGAGCCTCCACAGGAGACCCCACCCAGCAGCCCAGCCCCTACCCAGGAGGCCCCAGAGCTCAGGGCGCCTGGGTGGATTCTGAACAGCCCCGAGTCACGGTGGGTATAGTGGGAGCTACTACCACTGTGAGAAAAGCTATGTCCAAAACTGTCTCCCGGCCACTGCTGGAGGCCCAGCCAGAGAAGGGACCAGCCGCCCGAACATACGACCTTCCCAGACCTCATGACCCCCAGCACTTGGAGCTCCACAGTGTCCCCATTGGATGGTGAGGA CTCGAG ccc3′

Last, PCR amplication of a human genomic DNA fragment containing thehuman JH3 fragment with flanking genomic DNA was achieved using primerpair Seq-ID25 and Seq-ID26, containing sites for restriction enzymesSalI and XbaI/HindIII, respectively, as indicated (FIG. 11 a).

Seq-ID25: 5′-GGA GTCGAC CCCTGCCTGGGTCTCAGCCCGGGGGTCTGTG-3′                SalI Seq-ID26: 5′-TATA TCTAGA ATAT AAGCTTAGCCATCTTACCTGAAGAGACGGTGACC-3′                    XbaI    HindIII

This way a PCR amplicon of 239 bp length containing the germline JH3gene segment with flanking DNA was obtained (Seq-ID27).

Seq-ID27: 5′gga GTCGAC CCCTGCCTGGGTCTCAGCCCGGGGGTCTGTGTGGCTGGGGACAGGGACGCCGGCTGCCTCTGCTCTGTGCTTGGGCCATGTGACCCATTCGAGTGTCCTGCACGGGCACAGGTTTGTGTCTGGGCAGGAACAGGGACTGTGTCCCTGTGTGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACC GTCTCTTCAGGTAAGATGGCTAAGCTT atat TCTAGA tata3′

The three DNA fragments Seq-ID21, Seq-ID24 and Seq-ID27 have been clonedsequentially into a shuttle vector containing unique BamHI, NheI, XhoIand XbaI restriction enzyme sites, such that a cassette containing genesegments VH3.30, DH1.26 and JH3 could be assembled by sequentialligation of the DNA fragments via the compatible restriction enzymesites. Seq-ID21 was ligated as a BamHI-NheI fragment into the BamHI-NheIlinearised shuttle vector, then NheI-XhoI digested fragment Seq-ID24 wasligated into NheI-XhoI linearised shuttle vector, already containingSeq-ID21, and last, SalI-XbaI digested fragment Seq-ID27 was ligatedinto XhoI-XbaI linearised shuttle vector already containing clonedSeq-ID21 and Seq-ID24, thereby generating an artificialVH3.30-DH1.26-JH3 cassette in a shuttle vector (FIG. 11 a).

The entire “quasi-germline” cassette containing the artificiallyassembled VH3.30, DH1.26 and JH3 gene segments was then cloned into theretroviral vector MigR1 (Pear et al. 1998) already containing the codingregion for a human pH chain (Seq-ID28, see below) cloned into the uniqueBglII and HpaI sites of the MigR1 vector (construct MigR1-muH, FIG. 11b). A unique XhoI site separating the VH coding region from the constantpH chain coding region in MigR1-muH (highlighted in boldface print inthe middle of Seq-ID28) could be used to ligate the VH3.30-DH1.26-JH3cassette in-frame to the constant pH chain coding region, withoutaffecting the amino acid sequence at the transition form JH to theconstant coding region.

Seq-ID28: 5′ AGATCTACCATGGAGTTTGGGCTGAGCTGGGTTTTCCTTGTTGCGATTTTAGAAGGTGTCCAGTGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCCGGCAGGTCCCTGAGACTCTCCTGTGCGGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAATGGGTCTCAGCTATCACTTGGAATAGTGGTCACATAGACTATGCGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGATACGGCCGTATATTACTGTGCGAAAGTCTCGTACCTTAGCACCGCGTCCTCCCTTGACTATTGGGGCCAAGGTACCCTGGTCACCGT CTCGAG CGCTAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTCCTGTGAGAATTCCCCGTCGGATACGAGCAGCGTGGCCGTTGGCTGCCTCGCACAGGACTTCCTTCCCGACTCCATCACTTTCTCCTGGAAATACAAGAACAACTCTGACATCAGCAGCACCCGGGGCTTCCCATCAGTCCTGAGAGGGGGCAAGTACGCAGCCACCTCACAGGTGCTGCTGCCTTCCAAGGACGTCATGCAGGGCACAGACGAACACGTGGTGTGCAAAGTCCAGCACCCCAACGGCAACAAAGAAAAGAACGTGCCTCTTCCAGTGATTGCCGAGCTGCCTCCCAAAGTGAGCGTCTTCGTCCCACCCCGCGACGGCTTCTTCGGCAACCCCCGCAAGTCCAAGCTCATCTGCCAGGCCACGGGTTTCAGTCCCCGGCAGATTCAGGTGTCCTGGCTGCGCGAGGGGAAGCAGGTGGGGTCTGGCGTCACCACGGACCAGGTGCAGGCTGAGGCCAAAGAGTCTGGGCCCACGACCTACAAGGTGACCAGCACACTGACCATCAAAGAGAGCGACTGGCTCAGCCAGAGCATGTTCACCTGCCGCGTGGATCACAGGGGCCTGACCTTCCAGCAGAATGCGTCCTCCATGTGTGTCCCCGATCAAGACACAGCCATCCGGGTCTTCGCCATCCCCCCATCCTTTGCCAGCATCTTCCTCACCAAGTCCACCAAGTTGACCTGCCTGGTCACAGACCTGACCACCTATGACAGCGTGACCATCTCCTGGACCCGCCAGAATGGCGAAGCTGTGAAAACCCACACCAACATCTCCGAGAGCCACCCCAATGCCACTTTCAGCGCCGTGGGTGAGGCCAGCATCTGCGAGGATGACTGGAATTCCGGGGAGAGGTTCACGTGCACCGTGACCCACACAGACCTGCCCTCGCCACTGAAGCAGACCATCTCCCGGCCCAAGGGGGTGGCCCTGCACAGGCCCGATGTCTACTTGCTGCCACCAGCCCGGGAGCAGCTGAACCTGCGGGAGTCGGCCACCATCACGTGCCTGGTGACGGGCTTCTCTCCCGCGGACGTCTTCGTGCAGTGGATGCAGAGGGGGCAGCCCTTGTCCCCGGAGAAGTATGTGACCAGCGCCCCAATGCCTGAGCCCCAGGCCCCAGGCCGGTACTTCGCCCACAGCATCCTGACCGTGTCCGAAGAGGAATGGAACACGGGGGAGACCTACACCTGCGTGGTGGCCCATGAGGCCCTGCCCAACAGGGTCACCGAGAGGACCGTGGACAAGTCCACCGAGGGGGAGGTGAGCGCCGACGAGGAGGGCTTTGAGAACCTGTGGGCCACCGCCTCCACCTTCATCGTCCTCTTCCTCCTGAGCCTCTTCTACAGTACCACCGTCACCTTGTTCAAGGTGAAATGAGCGGCCGCTTTACGC GTTAAC 3′

BglII-HpaI restriction enzyme sites at the 5′ and 3′ ends of the insert,respectively, are also highlighted in boldface print and are underlined,and indicate the transition to the MigR1 vector backbone (Pear et al.1998).

In order to replace the V-coding region in Seq-ID28 contained in theMigR1 retroviral backbone, with the VH3.30-DH1.26-JH3 “quasi-germline”cassette, the ca. 1.1 kb VH3.30-DH1.26-JH3 fragment needed to bere-amplified by PCR using a BglII containing forward and a XhoIcontaining reverse primers Seq-ID29 and Seq-ID30, respectively (FIG. 11a).

Seq-ID29: 5′-GA AGATCT CACCATGGAGTTTG-3′                 BglIISeq-ID30: 5′-ATCTTACCT CTCGAG ACGGTGA-3′                        XhoI

The resulting ca. 1.1 kb PCR-fragment was digested with BglII and XhoIand was ligated into BglII-XhoI linarized pH-chain containing MigR1vector, thereby resulting in the V-D-J recombination competentretroviral expression vector pVDJ-muH-MigR1 (FIG. 11 b).

b) Demonstration of Bona Fide V(D)J Recombination Occurring inRetroviral V-D-J Vectors Generating Diverse Sequences Upon Transductionin Precursor B Cells.

As a proof-of-concept, to demonstrate that proper V(D)J recombinationcan occur in retroviral vectors containing V, D and J gene segments in“quasi-germline” configuration, the vector pVDJ-muH-MigR1 wasco-transduced into A-MuLV-transformed preB cell line 230-238 togetherwith a retroviral IgL chain expression vector, as described in Example1b. Only, if a V(D)J recombination event occurs on the pVDJ-muH-MigR1construct, that results in the in-frame rearrangement of the V, D and Jgene segments, can a full-length human IgM antibody be expressed on thecell surface of the double-transduced cells. Transduction efficiency ofthe pVDJ-muH-MigR1 vector can be monitored by co-expression of theIRES-coupled EGFP marker gene. As can be seen in FIG. 12( a), a verysmall population of 0.04% of cells that were transduced with at leastthe pVDJ-muH-MigR1 construct (efficiency of transduction was 44.7%),displayed detectable IgM expression on the cell surface, as measured byFACS-staining with an anti-kappa light chain antibody. Notably,practically no IgM expressing cells were detectable in the cellpopulation not transduced with the pVDJ-muH-MigR1 construct (lower rightquadrant in FIG. 12( a)), demonstrating the specificity of the staining.

The rare IgM expressing cells detectable in the upper right quadrant ofFIG. 12( a) have been sorted by preparative cell sorting using aFACS-Aria high speed cell sorter (BD, Franklin Lakes, N.J.) and havebeen expanded in tissue culture for 8 days, in order to expand the cellsfor characterization of retroviral integrants. The FACS profile for EGFPexpression (indicative of integrated pVDJ-muH-MigR1 construct) andsurface IgM after 8 days of expansion showed possibly few clonallyexpanded cells that displayed IgM on the cell surface and contained thepVDJ-muH-MigR1 construct (as measured by green fluorescence, FIG. 12(b)). Distinct cell populations in the upper right area of the FACS-plotof FIG. 12( b) have been sorted and genomic DNA has been prepared frompooled cell populations.

Genomic DNA was analyzed by a diagnostic PCR using primers binding inthe pVDJ-muH-MigR1 construct upstream of the VH and downstream of the JHregion. As expected, the diagnostic PCRs resulted in two discrete bandsof almost equal intensity, indicative of unrearranged V, D and J genesegments (ca. 1.2 kb fragment) and a smaller, ca. 0.5 kb fragment,indicative of V(D)J recombined gene segments (data not shown).Sequencing of the larger PCR band confirmed that this PCR ampliconrepresented the unrearranged V-D-J cassette, still in “quasi-germline”configuration. These unrearranged constructs were still detectable inIgM positive cells, if the cells were transduced with more than oneconstruct, of which not all might be accessible for V(D)J recombination.The smaller PCR amplicon did not result in a unique sequence uponsequencing of the PCR product and needed to be subcloned into the pSC-BPCR cloning vector for sequence analysis of individual PCR fragments.

From 6 plasmids analyzed, 2 contained identical bona-fide V(D)Jrecombination sequences, that showed all characteristic features ofsite-directed joining of V, D and J gene segments by V(D)Jrecombination, including nucleotide loss at the coding regions andaddition of non-templated sequences (N-regions) that are catalyzed bythe precursor lymphocyte specific enzyme terminal deoxynucleotidyltransferase (TdT) (FIG. 12( b)).

Already the two recovered sequences, represented by sequence of clone225 (FIG. 12 b), are firm evidence for the capacity of the V, D, and Jgene segment containing retroviral expression vectors to undergo V(D)Jrecombination in precursor B lymphocytes, as there is no otherexplanation of how these sequences, showing all signs of a bona fideV(D)J recombination event, could have been generated otherwise, even ifthe efficiency at this point appeared to be low. It is concluded thatupon increase of the efficiency of V(D)J recombination in the context ofretroviral transduction of precursor lymphocytes a diverse collection ofantibody sequences can be generated from a limited number of retroviralvectors containing V, D and J gene segments in “quasi-germline”configuration.

Example 5 Identification and Characterization of Suitable SelectorMurine B Cell Lines for Retrocyte Display

a) The expression of endogenous antibodies in B cell lines canpotentially hamper their utilization as selector cells in retrocytedisplay, as pairing of endogenous mouse immunoglobulin chains withrecombinant fully antibody chains could negatively affect theircell-surface display, or can lead to the expression of mixed human-mouseimmunoglobulins with undefinable binding specificities. Therefore, apanel of Abelson murine leukemia virus (A-MuLV) transformed murine pre-Bcell lines described in the literature, were examined for intracellularexpression of endogenous IgM heavy chains (pH) using anti-murine IgMheavy chain antibodies coupled to FITC (Southern Biotech). These cellsincluded the lines 40E1, 230-238, 204-1-8 (Alt et al., 1981), 18-81,18-81 subclone 8-11 (Rosenberg & Baltimore 1978), 63-12 (here calledFA-12) cells from RAG-2 deficient mice (Sinkai et al., 1992), and1624-5, 1624-6 from triple surrogate light chain knock-out mice (Shimizuet al., 2002). Cells were permeabilized using the Fix/Perm-kit (Caltech)following the manufacturer's instructions. As depicted in FIG. 14, celllines FA-12, 40E1, and 18-81 subclone 8-11, 1624-5 and 1624-6, showedthe least or no signal for intracellular IgM staining qualifying them assuitable selector cells for Retrocyte Display.b) As Retrocyte Display is based on retrovirus vector-mediated genetransfer, the panel of murine, A-MuLV transformed pre-B cell lines wasfurther examined for their susceptibility to retroviral transductionusing ecotropic MLV-derived vector particles containing a greenfluorescent protein (GFP) marker gene 9FIG. 13). 1×10⁵ cells weretransduced at an MOI of 0.5 using a vector preparation having packagedthe reporter gene GFP encompassing transfer vector LEGFP-N1 (Clontech)previously titrated on 18-81 subclone 8-11 pre-B cells by limitingdilution. Vector particles were generated as described below.Transduction was performed by spin-infection, essentially usingcentrifugation in 1.5 ml-Eppendorf tubes at 3.300 rpm and 30° C. for 3hours. Two days post transduction, gene transfer was analyzed bydetermining the frequency of CFP expressing cells by FACS-analysis.Untreated, naïve target cells served as negative controls. Asillustrated in FIG. 13, only original 18-81 cells showed very lowpermissiveness to MLV vector transduction (<10%). All other cell linesrevealed gene transfer efficiencies of 40%. Notably, with FA-12, 40E1and 1624-5 cells gene transfer efficiencies were maximal andreached >50% in the current experiment.

Taken together, FA12, 40E1 and 1624-5 cells were found to be bestsuitable for Retrocyte Display considering both criteria, a) low orabsent endogenous murine immunoglobulin expression and b) susceptibilityfor retroviral transduction. However, as it is desired to expressimmunoglobulins composed of recombinant heavy and light chains, B cellsare preferred that lack expression of surrogate light chain componentsas well (expressible from λ5, or VpreB1, or VpreB2 genes) as those mayalso compete for heavy chain association with recombinant light chainsin wild-type preB cells. Thus, 1624-5 cells derived from surrogate lightchain triple knockout mice are expected to be the best-suited cells forfurther retrocyte display technology. However, it shall be understood,that any other cell line, including the additional cell lines analyzedhere, which satisfy the criteria for no/low endogenous immunoglobulinexpression and retroviral transducibility, may be used to realize themethod disclosed herein.

Example 6 Generation of Selector Cells Clonally and Stably ExpressingFully Human Antibody Libraries

To generate vector particles having packaged transfer vectors encodingfully human antibody chains and libraries thereof and their subsequentemployment for the transduction of murine B cell lines, infectionexperiments were performed by the following method. Human embryonickidney 293T-HEK cells were plated at 2×10⁶ in 10 ml of DulbeccosModified Eagle Medium (DMEM), supplemented with 10% fetal calf serum(FCS) and L-Glutamin per 10 cm tissue culture dish, 16 to 24 hours priorto transfection. Mixtures of 5 μg of the respective transfer vectorsIgL(245)-LIB-IRES-YFP and IgH(650)-LIB-IRES-GFP (encoding libraries ofheavy or light chains linked by an IRES to GFP or YFP expression, FIG.15), 3 μg of pVPack-GP (an expression construct harboring gag and polgenes of MLV) and 2.5 μg of pVPack-Eco (an expression constructencompassing the env gene of ecotropic MLV, both STRATAGENE) wereprepared and incubated with 30 μl of Fugene (Roche) in 1 ml serum-freeDMEM, and were left standing for 15 to 30 minutes at room temperature.The Fugene/DNA mix was then gently added to the 293T-HEK cells seeded inthe 10 cm dishes. Heavy and light chain-encoding transfer vectors weretransfected into separate transient packaging cells.

48 hours post transfection, vector particle-containing supernatants werecollected from transient packaging cells and centrifuged at 3,000 rpm toremove contaminating cells. 1.5×10⁶ 1624-5 murine B cells were suspendedin 1 ml of media supplemented with different quantities of vectorparticles (diluted 1:1; 1:5; 1:20; 1:50; 1:100; 1:200) having packagedthe heavy or light chain-encoding regions of antibodies. Transductionwas performed by centrifugation in 1.5 ml-Eppendorf cups at 3.300 rpmand 30° C. for 3 hours. Unused supernatants were stored at −80° C. forutilization at a later time-point. To ensure that single copies oftransfer vectors integrated into the host cell genome, cells revealingfour days post infection gene transfer efficiency lower than 10%(detected by expression of GFP or YFP) were enriched using FACS (FIG.16). The cells were expanded for six days and subjected to a secondtransduction procedure employing previously frozen vector particleshaving packaged the light chain coding regions of antibodies at adilution of 1:5 as described above. Here, GFP-positive cells selectedfor heavy chain expression were infected with vector particlestransducing the light chain-IRES-YFP library and vice versa (FIGS. 15 &16). Four days post infection, successfully transduced cells expressingGFP and YFP were enriched using FACS. Approximately 20% of the cellsshowed GFP and YFP expression after the second transduction. To securethat only single vector integrations occurred per cell about one thirdof the populations were enriched that revealed only low or moderateexpression of the reporter gene transduced in the second round (approx.8%, see FIG. 16).

Example 7 Detection and Enrichment of Antigen-Reactive Human AntibodyExpressing Cells by Retrocyte Display

a) As a preparation for a Retrocyte Display proof of concept experiment,firstly the optimal staining and detection conditions for IL-15 bindingantibodies retrovirally expressed on selector cells was determined.1624-5 A-MuLV transformed preB cells co-expressing a human IgH and IgLchain library were mixed at a ratio of 2:1 with 1624-5 A-MuLVtransformed preB cells expressing retroviral expression vectors encodinga human anti-IL-15 antibody on the cell surface. Mixed cell samples wereincubated with various concentrations of recombinant human IL-15 (0.1 to2.5 μg/ml), and various concentrations of a polyclonal, biotinylatedanti-hu-IL-15 antibody (1.0 and 3.0 μg/ml), as indicated, which waseventually revealed with streptavidin-phycoerythrin (strep-PE). Todiscriminate cells displaying antibodies from non-immunoglobulinexpressing cells, samples were additionally counter-stained with ananti-hu-IgκL-APC antibody. As can be seen in the two upper-right FACSpanels of FIG. 17, IL-15 reactive cells were most efficiently detected(20.1 and 20.4%) using a combination of 0.1 and 0.5 μg/ml of recombinantIL-15 as a primary reagent, and 3.0 μg/ml of the biotinylatedanti-hu-IL-15 antibody as a secondary staining reagent.b) Next, a proof-of-concept experiment was performed, in which areference antibody specific for human IL-15 was spiked into a pool ofcells expressing a diverse library of human antibodies, upon which thespiked-in antigen-reactive cells have been analyzed by FACS. Inpreparation of this experiment, a library of antibodies retrovirallyexpressed in 1624-5 cells (see Example 6), was stained for surface Igexpression and for IL-15 binding (NC), alongside with a 1624-5 cell lineexpressing reference antibody specific for the human IL-15 antigen (PC).FACS analyses on these NC and PC cell lines are shown in FIG. 18, anddemonstrate the specific IL-15 staining of the anti-IL-15 referenceantibody displayed on the surface of the PC cells. In order to analyze,whether the reference anti-IL-15 Ab expressing cell line is stillquantitatively detectable by FACS, if the PC cells are spiked into theNC cell line expressing a random collection of human antibodies,different dilutions of PC cells in the NC library were analyzed by FACSfor specific IL-15 binding using the optimized IL-15 staining conditionsdetermined above. FACS-analysis of negative control cells revealed onlya small population exhibiting IL-15-binding activity. In contrast, morethan 60% of the positive control population was demonstrated to bindIL-15. Upon mixing both above populations at ratios of 10, 12.5, 25,37.5, and 50%, a correlation between the percentages of positive controlcells mixed into the antibody library cell pool with the fraction ofcells shown to bind IL-15 was observed. Thus, it is concluded that IL-15reactive cells can quantitatively be detected by FACS staining inmixtures with other non-specific antibody expressing cells.c) Next a proof of concept experiment was performed, in which rare IL-15reactive cells were enriched by Retrocyte Display. For this, a highlydiverse collection of human antibodies expressed in 1624-5 preB cellswas generated by retroviral transduction of an IL-15 IgH chain (coupledto GFP), and co-transduction of a complex collection (complexityapproximately 7×10⁴) of human IgκL chains (coupled to YFP). Thus, aRetrocyte Display antibody library was created by shuffling of a diversecollection of human IgκL chains against a single IgH chain from a humananti-IL-15 specific antibody. Cells were stained for IL-15 reactivityusing optimized conditions as determined before. IL-15 reactive cellswere enriched by three consecutive rounds of high-speed FACS cellsorting, followed by cell culture expansion. After three rounds ofRetrocyte Display enrichment, a cell population could be obtainedexpressing human antibodies and that essentially stained quantitativelyfor the antigen IL-15 from an initial cell population in which IL-15reactive cells were hardly detectable (FIG. 19). This experimentdemonstrated that repeated rounds of Retrocyte Display enrichment couldefficiently be used to enrich IL-15 binding cells.d) Confirmation of IL-15 binding specificity of individual cell clonesestablished from a 3×IL-15 enriched cell pool (see previous example).Next, from the cell pool which was 3 times subjected to IL-15-specificRetrocyte Display enrichment, 25 individual cell clones have beenestablished by single cell sorting. These 25 cell clones have beencharacterized for their IL-15 specificity by IL-15 staining, using thepreviously optimized staining conditions (see Example 7a). As a controlfor the specificity of the IL-15 staining, all clones were alsoincubated with all staining reagents, except for the IL-15 antigen. Themajority of the single cell clones displayed a highly specific IL-15staining pattern that was lost when the IL-15 antigen was left out ofthe staining reaction. Representative IL-15 specific stainings are shownin FIG. 19 with 4 selected individual cell clones. Stainings for theseclones are representative of altogether 25 cell clones (termed inalphabetical order A to Y) established from a 3×IL-15 antigen enrichedcell population, which all tested positive for IL-15 antigen binding.Negative and positive controls for the specificity of the stainings areprovided in FIG. 19, as indicated.

Example 8 Chain Shuffling or Guided Evolution Approach: Detection andIterative Enrichment of Antigen Specific Human Antibody Expressing Cellsby High-Speed Cell Sorting, Cloning of the Variable Region CodingRegions from Antigen Selected Cells and Confirmation ofAntigen-Specificity

As described above (Example 6), a cell library was generated expressinga library of human IgκL chains with a complexity of approximately1.2×10⁵ in combination with the heavy chain of reference antibodySK48-E26 (Young et al., WO 95/07997 A1) directed against the targetantigen human IL-1β. The retroviral vector backbone harbouring thesechains is depicted in more detail in FIGS. 4 c and 11 (see also Example4). For this, 3×10⁶ 1624-5 A-MuLV transformed 1624-5 preB cells weretransduced with the SK48-E26 IgH chain-encoding transfer vectorharbouring particles at a MOI less than 0.1. One day after transductionCFP-positive cells were enriched by standard high speed cell sortingusing a FACSAria from BD. Sorted cells were expanded in tissue culturein a humidified incubator for five days. Following expansion, the cellpopulation was transduced with particles having packaged the IgκL chainlibrary at a MOI of 1.5 by standard spin infection as described above(Example 5), and cells were allowed to recover from transduction for twodays in tissue culture. Following the two days recovery and expansionperiod, 5×10⁵ cells co-expressing GFP and YFP, and thus harbouring atleast one heavy and one light chain construct, were enriched usingpreparative cell sorting using a FACSAria from BD. The cell populationnow enriched for co-expression of IgH and IgL chain constructs wasexpanded for another four days in tissue culture. After this finalexpansion, an aliquot of 2×10⁶ cells expressing the IgH/IgL-chainlibrary were stained with 2 μg/ml in a volume of 100 μl with recombinanthuman IL-1β (R&D Systems) for 30 minutes on ice followed by two washingsteps using phosphate buffered saline (PBS) supplemented with 1% fetalcalf serum (FCS). After incubation with polyclonal antibodies directedagainst IL-1β and conjugated to biotin, cells were washed again twiceand subsequently stained with streptavidin-APC for detection ofantigen-binding cells and their subsequent enrichment using flowcytometry. After a first round of cell sorting by FACS, cells wereexpanded for five days and subjected to another round of anti-IL-1βstaining and enrichment of positively staining cells as described above.This selection was repeated three times (FIG. 21). The cell populationobtained after three rounds of Retrocyte Display enrichment, was againstained for IL-1β binding as described above, but this time reactivecells were not enriched as bulk populations as previously, butindividual cell clones were sorted into 96 well plates by means ofsingle cell sorting using a FACSAria from BD. Following seven days ofcultivation and expansion, individual cell clones were again analysedfor IL-1β antigen-specificity using the described protocol and, inaddition, as a negative control, with all secondary reagents, except theantigen IL-1β. As expected and demonstrated using flow cytometry, someclones showed specific binding to the target antigen as revealed by aspecific FACS signal in the presence of the antigen, but not in theabsence of it (excluding background binding of the clones to any of thesecondary detection reagents). However, some clones showed a stainingsignal irrespective of the presence of the antigen indicating that theseclones non-specifically bound to any of the secondary reagents used forthe detection of IL-1β reactivity. In total, genomic DNA of 24 cellclones was isolated and served as a template for standard genomic PCRemploying oligonucleotides Seq-ID31 and Seq-ID32, specifically bindingup- and down-stream of the variable region of human light chains encodedin the retroviral light chain library, respectively.

Seq-ID31: 5′-CCTTGAACCTCCTCGTTCGACCC-3′Seq-ID32: 5′-AGGCACAACAGAGGCAGTTCCAG-3′

PCR amplicons of expected size were obtained from each analyzed cellclone, and the PCR amplicons were directly subjected to DNA sequenceanalysis. Of the 24 clones analysed twelve were shown to harbour anidentical, but novel IgκL chain, termed LCB24, and, as expected alsoharboured the IgH chain of SK48-E26, as determined separately.

As expected, all 12 clones expressing the LCB24 IgL chain in combinationwith the SK48-E26 IgH chain displayed specific IL-1β-signals using flowcytometry as mentioned above.

A selected PCR amplicon containing the amplified novel LCB24 IgκL chainwas digested with the restriction enzymes HindIII and Eco47III flankingthe variable coding region (FIG. 4 c), and the fragment was cloned intoa retroviral IgκL chain expression vector with compatible restrictionenzyme sites allowing the in-frame fusion of the LCB24 V_(L) codingregion to the constant human kappa light chain coding region. Thus, theresultant vector encoded a novel, fully human IgκL chain.

The re-cloned, and sequence verified retroviral expression vector forthe LCB24 Ig L chain was transduced together with the SK48E26 IgH chainof the IL-1β reference antibody SK48-E26 into 1624-5 cells. Afterexpansion in tissue culture for 2 days, GFP+/YFP+ cells were enriched byhigh-speed cell sorting using a FACSAria from BD. The resulting, Igexpressing cells were first tested for their ability to bind IL-1β asdescribed. As expected their reactivity mediated by display of LCB24together with the heavy chain of SK48-E26 was confirmed (FIG. 22). Toexclude that the novel antibody was generally cross-reactive to otherantigens or proteins, the cells expressing the LCB24 IgL/SK48-E26 IgHcombination were assayed for IL-15 reactivity, as described before. Asdepicted in FIG. 23, no reactivity for IL-15 could be detected for thenovel IgL LCB24/HC SK48-E26 antibody, indicating the targetantigen-specificity of the novel antibody. Further controls included acell line expressing an anti-IL-15 specific reference antibody (aspositive control) and the original SK48-E26 IL-1β antibody. While theanti-IL-15 antibody expressing cells, as expected, showed specificstaining to IL-15, no reactivity was detected for the SK48-E26 IL-1βantibody or for cells (FIG. 23).

In summary, a novel light chain mediating antigen-specific reactivitywas identified in a screening experiment employing a library of lightchains shuffled against the heavy chain of an IL-1β specific referenceantibody SK48-E26.

Example 9 Retrocyte Display Screening on Shuffled IgH and IgL ChainLibraries. Detection and Iterative Enrichment of Antigen Specific HumanAntibody Expressing Cells by High-Speed Cell Sorting, Cloning of theVariable Region Coding Regions from Antigen Selected Cells andConfirmation of Antigen-Specificity

As described above (Example 6), a cell library was generated expressinga library of heavy chains with a complexity of approximately 6.5×10⁵(coupled to GFP) using a MOI of approximately 0.1. The retroviral vectorbackbone harbouring these chains is depicted in FIGS. 4 c and 11 in moredetail (see also Example 4). For this, 3×10⁶ 1624-5 A-MuLV transformed1624-5 preB cells were transduced with the above-mentioned IgHchain-library encoding transfer vector harbouring particles at a MOIless than 0.1. Two days after transduction, GFP-positive cells wereenriched by standard high speed cell sorting using a FACSAria from BD.After sorting of GFP+ cells, the cells were expanded in tissue culturefor two additional days. After expansion, the GFP+ cell population wastransduced with particles having packaged a light chain libraryconsisting of 245 fully sequence characterized light chains at a MOI >1as described before. Two days post transduction, GFP+/YFP+ doubletransduced cells were enriched by high speed cell sorting and the cellpopulation, now harbouring both IgH and IgL chain libraries in themajority of the cells were again expanded for three days in tissueculture. After this, an aliquot of 2.5×10⁵ cells was stained with acocktail of antigens including inter alia SAV (streptavidin)-APC-Cy7 asdescribed above (see Example 8), and enriched for reactivity of thetarget antigen using flow cytometry. In parallel, the cell populationexpressing the antibody IgH/IgL library was stained using anti-IgL kappaspecific antibodies. Approximately 75% of these cells were found todisplay human antibodies on the cell surface (data not shown).Antigen-reactive cells have been sorted by high-speed cell sorting usinga FACSAria from BD, and enriched cells were expanded in tissue culturefor seven days. The same staining and cell enrichment procedures, asdescribed above were repeated twice more. After three Retrocyte Displayselection rounds the resultant cell population was again stained toassess binding of the target antigen SAV-APC-Cy7 and analysed using flowcytometry. As depicted in FIG. 24, the bulk population obtained showedbinding to SAV-APC-Cy7 indicating the successful selection of antibodieswith antigen-specificity to SAV-APC-Cy7. To assess the specificity ofreactivity against the target antigen SAV-APC-Cy7 the three-timesenriched library expressing cells were also stained with the antigensSAV-APC and SAV-PerCP-Cy5.5 (FIG. 25). Similar to un-transduced cellsand unselected library expressing cells serving as negative controls,these antigens were not bound by the three-times SAV-APC-Cy7 enrichedcells. However, the later cells did again reveal strong reactivity toSAV-APC-Cy7, indicating that the antigen-specificity of the selectedcell population was directed against the Cy7 fluorochrome of theSAV-APC-Cy7 tandem dye.

Genomic DNA of the 3-times enriched cell population was isolated andserved as a template for standard genomic PCR employing oligonucleotidesSeq-ID31 (see above) and Seq-ID33 specifically binding up- anddown-stream of the coding regions of human light and heavy chainsencoded in the retroviral libraries.

Seq-ID33: 5′-CGGTTCGGGGAAGTAGTCCTTGAC-3′)

PCR amplicons for the heavy chains and light chains of expected sizewere obtained and were separately subcloned into a standard PCR-fragmentcloning vector pSC-B (Stratagene), as recommended by the manufacturer.pSC-B plasmid clones harbouring the cloned heavy and light chain regionswere isolated from 10 bacterial clones each resulting from the IgH PCRfragment subcloning and the IgL PCR fragment subcloning, which were allsubjected to DNA sequence analysis. DNA sequencing revealed twodifferent IgH chain sequences termed HC49, HC58 and two different IgLchain sequences termed LC4 and LC10.

DNA fragments containing the V_(H) and V_(L) coding regions wereisolated from sequence verified clones by HindIII and Eco47IIIdigestion, as these restriction enzyme sites flank the variable regions(see FIG. 4 c) of the respective V_(H) and V_(L) regions of HC49, HC58,LC4 and LC10. The isolated V_(H) and V_(L) regions of HC49, HC58, LC4and LC10 were cloned into a retroviral recipient vector harbouring theconstant regions of a human Igγ1H chain (expression IRES coupled to GFP)and a human IgκL chain (expression IRES coupled to YFP), respectively,as described above. Thus, retroviral expression vector constructs weregenerated encoding fully human IgH and IgL chains for the novel HC49 andHC58 IgH chains, LC4 and LC10 IgL chains. Upon co-transduction of thesevectors into 1624-5 preB cells and expansion for 8 days, GFP+/YFP+ cellswere enriched using flow cytometry as before. Resultant cells were firsttested for their ability to bind SAV-APC-Cy7 as described. As depictedin FIG. 26, reactivity of cells expressing the antibodies HC49/LC4 andHC/LC10 did not show significant binding activity against SAV-APC-Cy7.In contrast, reactivity mediated by antibodies HC58/LC4 and HC58/LC10was readily detected. This provides a proof of concept for thesuccessful identification of novel antigen-specific antibodies byRetrocyte Display based on the shuffling of diverse collections of IgHchains and IgL chains, without the need of either a known IgH or IgLchain from a reference antibody with known antigen-specificity.

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The invention claimed is:
 1. A plurality of stably transduced vertebrate host cells, wherein each member of the plurality of stably transduced vertebrate host cells (i) comprises a stably integrated retroviral expression vector encoding a full-length antibody heavy chain polypeptide, a stably integrated retroviral expression vector encoding a full-length antibody light chain polypeptide, and a screening marker, and (ii) expresses on the cell surface a full-length antibody that is encoded by the stably integrated retroviral expression vectors, wherein each member of the plurality of stably transduced vertebrate host cells comprises a different full-length antibody heavy chain polypeptide or a different full-length antibody light chain polypeptide, and wherein the plurality of stably transduced vertebrate host cells is generated by: (a) transducing a first library of replication incompetent retroviral expression constructs encoding a plurality of full-length antibody heavy chain or light chain polypeptides into a population of vertebrate host cells such that a first library of stably transduced vertebrate host cells is generated, wherein the first library of replication incompetent retroviral expression constructs comprise a murine stem cell virus (MSCV) long terminal repeat (LTR), wherein the retroviral transduction is performed at a multiplicity of infection of equal to or less than 0.1, wherein each member of the first library of stably transduced cells comprises at least one retroviral expression construct from the first library of retroviral expression constructs stably integrated into the host cell genome and wherein the vertebrate host cells are Abelson-murine leukemia virus transformed precursor B lymphocytes that do not express lamda-5, VpreB1, and VpreB2 surrogate light chain genes; (b) transducing the first library of stably transduced cells of step (a) with a second library of replication incompetent retroviral expression constructs encoding a plurality of full-length antibody heavy chain or light chain polypeptides such that a second library of stably transduced vertebrate host cells is generated, wherein the first library of retroviral expression constructs encodes a plurality of full-length antibody heavy chain polypeptides and the second library of retroviral expression constructs encodes a plurality of full-length antibody light chain polypeptides, or the first library of retroviral expression constructs encodes a plurality of full-length antibody light chain polypeptides and the second library of retroviral expression constructs encodes a plurality of full-length antibody heavy chain polypeptides such that the first and the second library of retroviral expression constructs encode antibody heavy and light chain combinations, wherein the first or the second library of retroviral expression constructs further comprise at least one screening marker which is a fluorescent protein or cell surface marker, wherein the second library of replication incompetent retroviral expression constructs comprise a murine stem cell virus (MSCV) long terminal repeat (LTR), wherein the retroviral transduction is performed at a multiplicity of infection of equal to or less than 0.1, and wherein each member of the second library of stably transduced cells comprises at least one retroviral expression construct from the first library of retroviral expression constructs stably integrated into the host cell genome and at least one retroviral expression construct from the second library of retroviral expression constructs stably integrated into the host cell genome; and (c) allowing expression of the first and second stably integrated retroviral expression constructs in the second library of stably transduced vertebrate host cells such that full-length antibodies are expressed on the surface of the second library of stably transduced vertebrate host cells, thereby producing the plurality of stably transduced vertebrate host cells.
 2. The plurality of stably transduced vertebrate host cells of claim 1, wherein the full-length antibody is selected from the group consisting of a fully human antibody, a humanized antibody, and a chimeric antibody.
 3. The plurality of stably transduced vertebrate host cells of claim 1, wherein the population of vertebrate host cells are derived from a cartilaginous fish, bony fish, amphibian, reptile, bird, or mammal.
 4. The plurality of stably transduced vertebrate host cells of claim 3, wherein the mammal is a mouse, rat, rabbit, guinea pig.
 5. The plurality of stably transduced vertebrate host cells of claim 4, wherein the mammal is a mouse.
 6. The plurality of stably transduced vertebrate host cells of claim 1, wherein the second library of vertebrate host cells ectopically express Igα and Igβ molecules.
 7. The plurality of stably transduced vertebrate host cells of claim 1, which has a complexity of at least 6×10⁵.
 8. The plurality of stably transduced vertebrate host cells of claim 1, wherein the fluorescent protein is selected from the group consisting of green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP) and blue fluorescent protein (BFP).
 9. The plurality of stably transduced vertebrate host cells of claim 1, wherein the cell surface marker is selected from the group consisting of CD7, CD34 and the low affinity nerve growth factor receptor.
 10. The plurality of stably transduced vertebrate host cells of claim 1, 8 or 9, wherein the first and second library of retroviral expression constructs comprise different screening markers.
 11. The plurality of stably transduced vertebrate host cells of claim 1, 8 or 9, wherein the at least one screening marker is operably linked to the nucleotide sequence encoding the full-length antibody heavy chain polypeptide or the full-length antibody light chain polypeptide using at least one internal ribosomal entry sequence (IRES).
 12. The plurality of stably transduced vertebrate host cells of claim 1, wherein the first or second library of retroviral expression constructs further comprise at least one antibiotic selection marker.
 13. The plurality of plurality transduced vertebrate host cells of claim 12, wherein the first and second library of retroviral expression constructs comprise different antibiotic selection markers.
 14. The plurality of plurality transduced vertebrate host cells of claim 12 or 13, wherein the at least one antibiotic selection marker is operably linked to the nucleotide sequence encoding the full-length antibody heavy chain polypeptide or the full-length antibody light chain polypeptide using at least one internal ribosomal entry sequence (IRES). 